Methods for detecting enveloped virus infections by measuring cell surface TSG101

ABSTRACT

The invention involves the detection of virally infected cells by antibodies or antibody fragments which selectively bind to TSG101. TSG101 is on the surface of mammalian cells, and thus available for detection by antibodies, during viral budding—a phenomenon wherein viral particles escape a virally infected cell after propagation in that cell, so as to infect other cells. To achieve budding, a protein, TSG101 is “hijacked” and misdirected to, or mis-expressed on, the surface of the infected cell. Antibodies can be used to selectively detect such infected cells. Certain TSG101 antibodies may provide therapeutic benefit when administered to infected mammals.

PRIORITY DATA AND INCORPORATION BY REFERENCE

This application is a continuation-in-part of application Ser. No.10/675,979, filed Oct. 1, 2003, now U.S. Pat. No. 7,427,468 which claimsthe benefit under 35 U.S.C. § 119 (e) of U.S. Provisional PatentApplication No. 60/415,299, filed on Oct. 1, 2002, which is incorporatedby reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies that binds a TSG101 proteinand inhibit or reduce viral production. The invention also relates tomethod of using the TSG101 antibodies for the treatment of viralinfections, including HIV infection. The invention further relates tomethods of detecting viral infected cells using TSG101 antibodies.

BACKGROUND OF THE INVENTION

Pathogen and host cell interactions play critical roles in thepathogenesis of viral diseases such as AIDS. For a typical viralinfection, viruses have to attach to the host cells through cell surfacereceptors, fuse with host cell membrane, translocate across the cellmembrane, uncoat viral particles, synthesize and assemble viral proteinsusing host protein synthesis machinery, and release from host cellsthrough host exporting machinery. The interplay between the viruses andhost cells determine the outcome of viral pathogenesis, ranging from theelimination of viruses to a parasitic or lethal infection. For example,HIV employs a variety of strategies to productively infect human cells.A retrovirus, its life cycle begins by attaching to host cells—theprimary target is the CD4+ T helper cells and gaining entry via specificreceptors. In the cell, the RNA genome is “reverse” transcribed to itscomplementary DNA, and then shuttled to the nucleus for its integrationin the host genome. This integrated “provirus” then directs theproduction of new viral RNA and proteins, which self-assemble and then“bud” from the cell as mature—and infections—viral particles, envelopedin plasma membrane. Like all viruses, the HIV is a parasite, unable tocatalyze the membrane fission event that drives the budding process.Instead, the nascent virus recruits the cell's membrane sortingmachinery to complete this final stage of infection. Such an host andvirus interplay has been well demonstrated in individuals, who carry adefective cell surface receptor (CCR5), are completely resistant to HIVinfection, elucidating the important roles of host genes and geneticpathways in viral pathogenesis.

Tumor Susceptibility Gene 101 (TSG101, Li et al., 1996, Cell, 85,319-29) plays important roles in cell growth (Zhong et al., 1998, CancerRes. 58, 2699-702, Oh et al., 2002, Proc Natl Acad Sci U.S.A 99, 5430-5;Krempler et al., 2002, J. Biol Chem 277, 43216-23; Wagner et al., 2003,Mol. Cell. Biol 23, 150-62; LI et al., 1996, Cell 85, 319-29), cellularprotein trafficking (Babst et al., 2000, Traffic 1, 248-58; Bishop etal., 2002, J. Cell Biol. 157, 91-101), and degradation of p53 (Li etal., 2001, proc Natl Acad Sci U.S.A 98, 1619-24; Ruland et al., 2001,Proc Natl Acad Sci U.S.A 98, 1859-64; Moyret-Lalle et al., 2001, CancerRes 61, 486-8). TSG101 is also widely recognized as a key player in thisfinal stage, inhibition of cellular TSG101 blocks the budding process ofHIV. Acting in concert with other cellular factors, TSG101 thus plays anessential role in the budding or spread of HIV viruses. The HIV Gagprotein, previously shown to orchestrate viral assembly and budding,binds with high affinity to TSG101, and this Gag/TSG101 interaction isessential for efficient HIV viral assembling and budding, as disruptionof the Gag/TSG101 interaction prevents HIV viral budding, andsignificantly limit the spread of HIV virus.

The final step in the assembly of an enveloped virus assembly requiresseparation of budding particles from the cellular membranes. Threedistinct function domains in Gag, i.e., PTAP in HIV-1 SEQ ID NO.: 7)(Gottlinger et al., 1991,. Proc Natl. Acad. Sci. U.S. A 88, 3195-9;Huang et al., 1995, J. Virol 69, 610-8); PPPY in RSV (SED IS NO.: 8)(Parent et al. 1995, J. Virol 69, 5455-60), MuLV (Yuan et al., 1999,Embo J 18, 4700-10), and M-PMV (Yasuda et al., 1998, J. Virol 72,4095-103); and YXXL in EIAV SEQ ID NO.: 6) (Puffer et al., 1997, J.Virol 71, 6541-6), have been identified in different retroviruses thatare required for this function and have been termed late, or L domains(Wills et al., 1991, Aids 5, 639-54). In HIV-1, the L domain contains aPTAP motif and is required for efficient HIV-1 release (see, e.g., Willset al., 1994, J. Virol. 68, 6605-6618; Gottlinger et al., 1991, Proc.Natl. Acad. Sci. USA 88, 3195-3199, Huang et al., 1995, J. Virol. 69,6810-6818). The L domain of HIV-1 p6, especially the PTAP motif, bindsto the cellular protein TSG101 and recruits it to the site of virusassembly to promote virus budding (VerPlant et al., 2001, Proc. Natl.Acad. Sci. USA 98: 7724-7729; Garrus et al., 2001, Cell 107:55-65;Martin-Serrano et. Al., 2001, Nature Medicine 7:1313-19; Pornillos etal., 2002, EMBO J. 21:2397-2406; Demirov et al., 2002, Proc Natl. Acad.Sci. USA 99:955-960; PCT Patent Publication WO 02/072790; U.S. PatentApplication Publication No. U.S. 2002/0177207). The UEV domain of TSG101binds the PTAP motif and mono-ubiquitin (Pronillos et al., 2002, EmboJ21, 2397-406; Pornillos et al., 2002, Nat. Struct Biol. 9, 812-7),which has also been implicated in HIV-1 budding (Patnaik et al., 2000,Proc. Natl. Acad. Sci. U.S. A 97, 13069-74; Schubert et al., 2000, ProcNatl. Acad. Sci. U.S. A 97, 13057-62; Strack et al., 2000, Proc Natl.Acad. Sci. U.S. A 97, 13063-8). Depletion of cellular TSG101 (Garrus etal., 20001, Cell 107:55-65) or over-expression of a truncated form ofTSG101 inhibits HIV-1 release (Demirov et al., 20002, Proc. Natl. Acad.Sci. USA 99-955-960). Under certain circumstances, TSG101 can evensubstitute for the HIV-1 L domain topromote virus release(Martin-Serrano et al., 2001, Nature Medicine 7:1313-19).

In yeast, the tag 101 ortholog Vps23 has been shown to interact withVps28 and Vps37 and to form a protein complex named ESCRT-1, which iscritical for endosomal protein sorting into the multivesicular bodypathway (Katzmann et al., 2001, Cell 106, 145-55). It is hypothesizedthat this intracellular multivesicular body formation resembles HIV-1release at the plasma membrane (Garrus et al., 2001, Cell 107:55-65;Patnaik et al., 2000, Proc. Natl. Acad. Sci. U.S.A 97, 13069-74). Inmammalian cells, TSG101 interacts with Vps28 to form an ESCRT-I-likecomplex (Babst et al., 2000, Traffic 1, 248-58; Bishop et al., 2002, J.Cell Biol. 157, 91-101; Bishop et al., 2001, J. Biol. Chem. 276,11735-42), although the mammalian homolog of Vps37 has not beenidentified.

Recent studies (Blower et al., 2003, AIDS Rev. 5:113-25; Vadiserri etal., 2003, Nat. Med. 9:881-6) have estimated that s many as 42 millionpeople worldwide have been infected with HIV. The disease has killedmore than 3 million people. While the advent of highly potent andtargeted combination therapies has slowed the progression of AIDS inindustrialized nations, the AIDS pandemic is causing a “humandevelopment catastrophe” in developing nations, particularly in Africa,where more than 21 million Africans have been infected. In South Africaalone, the death toll is projected to rise to 10 million by 2015.Related statistics portend a similar crisis in the Asia Pacific region,which, according to United nations' estimates, has more than 7 millionHIV-infected individuals. Repercussions from the AIDS pandemic extendwell beyond the clinic, which lack the resources to treat the swellingnumber of recently infected patients (nearly 20% of the adult populationin South Africa is infected). Treatment of HIV-infected and gravely illAIDS patients is stressing the already over-burdened health care systemsof Africa and other developing nations. Rose yet, current treatments ofHIV—despite their initial success in reducing viral load are beginningto lose their efficacy, as drug-resistant HIV strains are increasinglyisolated in newly infected individuals. Further compounding thetherapeutic management of HIV disease is the toxicity of currentantiretroviral regimens, the magnitude of which complicates thephysician's decision to begin and to maintain treatment. Identifying newtherapeutic paradigms for the treatment of HIV disease, especially thosewith mechanisms of action that promise to slow the development ofresistance, is indeed a global challenge for the biopharmaceuticalindustry.

Many viruses are also highly mutable. Methods and compositions relyingon targeting such viruses directly are normally not sufficient in thetreatment of infections by such viruses. For example, HIV-1 is a highlymutable virus that during the course of HIV-1 infection, the antibodiesgenerated in an infected individual do not provide permanent protectiveeffect due in part to the rapid emergence of neutralization escapevariants (Thali et al., 1992, J. Acquired Immune Deficiency Syndromes5:5911-599). Current therapies for the treatment of HIV-infectedindividuals focus primarily on viral enzymes involved in two distinctstages of HIV infection, the replication of the viral genome and thematuration of viral proteins. Since the virus frequently mutates,strains resistant to an antiviral inhibitor develop quickly, despite thedrug's initial therapeutic effects. In one recent study, the percentageof individuals newly infected with drug-resistant HIV strains increasedsix fold over a five year period (Little et al., 2002, N. Engl. J. Med.347:385-94). Further combination therapy, the current standard of carethat attacks HIV with inhibitors of both reverse transcriptase andprotease, is leading to the development of multi-drug resistant HIVstrains. Antiretroviral drugs directed against new HIV-based targets,while of considerable value, do not address this increasingly criticalissue. For example, HIV strains resistant to Fuzeon® (enfuvirtide), thenewest addition to the anti-HIV armamentarium, have already beenisolated from patients. Thus, despite its antiviral potency and novelmechanism of action, drug-resistance is likely to undermine thetherapeutic potential of viral fusion inhibitors, like Fuzeon®. There istherefore a need for developing novel therapeutics and preventativemeasure to combat viral infections such as HIV infection.

Discussion or citation of a reference herein shall not be construed asan admission that such reference is prior art to the present invention.

SUMMARY OF THE INVENTION

The present invention provides methods of using antibodies that bind aTSG101 protein to inhibit or reduce viral production. The presentinvention also provides methods of using TSG101 antibodies for treatingviral infections. The present invention also provides methods andcompositions for treating viral infection by targeting TSG101 on thesurface of infected cells, e.g., delivering therapeutic and/ordiagnostic agents, to such infected cells.

In one aspect, the invention provides a method for reducing viralbudding from a mammalian cell infected by an enveloped virus. The methodcomprises contacting said mammalian cell with a sufficient amount of anantibody that binds a TSG101 protein. In another aspect, the inventionprovides a method for delivering a therapeutic molecule to a mammaliancell infected by an enveloped virus, comprising contacting saidmammalian cell with an antibody conjugate comprising an antibody thatbinds a TSG101 protein conjugated with said therapeutic molecule. In apreferred embodiment, said antibody binds the N-terminal or C-terminalregion of said TSG101 protein. In a preferred embodiment, said mammaliancell is a human cell. IN another preferred embodiment, said antibodybinds an epitope comprises in the amino acid region selected from thegroup consisting of VRETVNVITLYKDLKPPVL (SEQ ID NO: 2) andQLRALMQKARKTAGLSDLY (SEQ ID NO: 3). Preferably, said antibody is amonoclonal antibody. In still another preferred embodiment, saidenveloped virus is selected from the group consisting of humanimmunodeficiency virus type I (HIV-I), human immunodeficiency virus typeII (HIV-II), Marburg virus, and Ebola virus.

In another aspect, the invention provides a method for treatinginfection by an enveloped virus in a mammal, comprising administering tosaid mammal a therapeutically effective amount of an antibody that bindsa TSG101 protein. In still another aspect, the invention provides amethod for treating infection by an enveloped virus in a mammal,comprising administering to said mammal a therapeutically effectiveamount of an antibody conjugate comprising an antibody that binds aTSG101 protein conjugated with a therapeutic agent. In a preferredembodiment, said antibody binds the N-terminal or C-terminal region ofsaid TSG101 protein. In a preferred embodiment, said mammal is a human.In another preferred embodiment, said antibody binds an epitopecomprises in the amino acid region selected from the group consisting ofVRETVNVITLYKDLKPVL (SEQ ID NO: 2) and QLRALMQKARKTAGLSDLY (SEQ ID NO:3). Preferably, said antibody is a monoclonal antibody. In still anotherpreferred embodiment, said enveloped virus is selected from the groupconsisting of human immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), Marburg virus, and Ebola virus.In one embodiment, the method further comprises administering to saidmammal a therapeutically effective amount of one or more othertherapeutic agents.

In still another aspect, the invention provides a method for identifyinga mammalian cell infected by an enveloped virus, comprising (a)contacting cells of a mammal with an antibody conjugate comprising anantibody that binds a TSG101 protein conjugated with a label; and (b)detecting a cell having said label attached, thereby identifying saidcell infected by said enveloped virus. In a preferred embodiment, saidantibody binds the N-terminal or C-terminal region of said TSG101protein. In a preferred embodiment, said mammalian cell is a human cell.In another preferred embodiment, said antibody binds an epitopecomprised in the amino acid region selected from the group consisting ofVRETVNVITLYKDLKPVL (SEQ ID NO: 2) and QLRALMQKARKTAGLSDLY (SEQ ID NO:3). Preferably, said antibody is a monoclonal antibody. In anotherpreferred embodiment, said enveloped virus is selected from the groupconsisting of human immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), Margurg virus, and Ebola virus.In one embodiment, said label is a fluorescence label, and said cellhaving said label attached is detected using a fluorescence activatedcell sorter.

The invention also provides a method for ex vivo removal of cellsinfected by an enveloped virus from a fluid derived from a mammal. Themethod comprises (a) incubating said fluid with a sufficient amount of aTSG101 antibody that binds a TSG101 protein; and (b) removing cellsbound by said TSG101 antibody from said fluid. Said fluid can be bloodor serum. In a preferred embodiment, said antibody binds the N-terminalor C-terminal region of said TSG101 protein. In a preferred embodiment,said mammal is a human. In another preferred embodiment, said antibodybinds an epitope comprise in the amino acid region selected from thegroup consisting of VRETVNVITLYKDLKPVL (SEQ ID NO: 2) andQLRALMQKARKTAGLSDLY (SEQ ID NO: 3). Preferably, said antibody is amonoclonal antibody. In another preferred embodiment, said envelopedvirus is selected from the group consisting of human immunodeficiencyvirus type I (HIV-I), human immunodeficiency virus type II (HIV-II),Marburg virus, and Ebola virus. In one embodiment, said cells bound bysaid TSG101 antibody are removed using a column comprising an antibodythat binds said TSG101 antibody.

The invention also provides a method for treating or preventinginfections by an enveloped virus in a mammal, comprising administeringto said mammal a therapeutically or prophylactically sufficient amountof a vaccine composition, wherein said vaccine composition comprises apolypeptide comprising a TSG101 protein. In a preferred embodiment, saidpolypeptide comprises an N-terminal or C-terminal region of said TSG101protein. In a preferred embodiment, said mammal is a human. In anotherpreferred embodiment, said polypeptide comprises an amino acid regionselected from the group consisting of VRETVNVITLYKDLKPVL (SEQ ID NO: 2)and QLRALMQKARKTAGLSDLY (SEQ ID NO: 3). In still another preferredembodiment, said enveloped virus is selected from the group consistingof human immunodeficiency virus type I (HIV-I), human immunodeficiencyvirus type II (HIV-II), Marburg virus, and Ebola virus. In oneembodiment, the method further comprises administering to said mammal atherapeutically effective amount of one or more other therapeuticagents.

The invention also provides a method for treating or preventinginfection by an enveloped virus in a mammal, comprising administering tosaid mammal a therapeutically or prophylactically sufficient amount of aDNA vaccine composition, wherein said DNA vaccine composition comprisesa polynucleotide molecule encoding a polypeptide comprising a fragmentof a TSG101 protein. In one embodiment, said polynucleotide moleculeencodes a polypeptide comprising an N-terminal or c-terminal region ofsaid TSG101 protein. In a preferred embodiment, said mammal is a human.In still another preferred embodiment, said polynucleotide moleculeencodes a polypeptide comprising amino acid sequence selected from thegroup consisting of VRETVNVITLYKDLKPVL (SEQ ID NO: 2) andQLRALMQKARKTAGLSDLY (SEQ ID No: 3), or a fragment of at least 5 aminoacids thereof. In still another preferred embodiment, said envelopedvirus is selected from the group consisting of human immunodeficiencyvirus type I (HIV-I), human immunodeficiency virus type II (HIV-II),Marburg virus, and Ebola virus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the 390 amino acid sequence of human TSG101 protein (SEQID NO: 1). (GenBank Accession No. U82130.1/GI: 1772663).

FIG. 2 depicts the effects of anti-TSG101 antibodies on MLV virusproduction. Left top panel: phoenix helper cells without treatment ofantibody (positive control) showed efficient production of retroviruses,and infection of N2A target cells; left middle panel: Rabbit IgG had noeffect; left bottom panel: a rabbit antibody against N-terminal TSG101had an effect of less than 20% inhibition; right top panel: a rabbitantibody against c-terminal TSG11 significantly inhibited the productionof retroviruses, and infection of N2A target cells (50-70% inhibition);right middle panel: a mixture of anti-C terminal and anti-N terminalantibodies gave similar results as the anti-C terminal antibody alone;right bottom panel: N2a cells that were not infected by viruses onlyshowed minimal background staining.

FIGS. 3A-E: GFP-TSG101 localizes to cell surface during virla release.Live confocal images of Phoenix helper cells with active viral release24 hrs after transfection of GFP-TSG101. 3A. Bright field images of fourcells; 3B-E. Live confocal fluorescence images of the same field atdifferent sections; White arrows point to cell surface localization ofGFP-TSG101.

FIG. 4: Cell Surface Localization of TSG101 during HIV duding. H9ΔBg1cells (CD4+ human T lymphocytes, carrying HIV viral integration) wereactively producing an releasing HIV virions with a defective envelopeprotein (this non-infectious form of HIV viruses will not infectionother cells, thus specifically allowing the study viral release) Theparental H9 cells that do not carry HIV were used as a control. BothH9ΔBg1 and H9 cells were fixed with 2% paraformoldehyde for 10 min atroom temperature (this surface fixation does not permeabilize cells).Anti-TSG101 antibody were incubated with both cells lines for 20 min anddetected with a fluorescence labels secondary antibody. Top panels:fluorescence images; Bottom panels: bright field images.

FIG. 5: FACS Profile of Cell Surface Localization of TSG101 during HIVBudding. Both H9ΔBg1 and H9 cells were fixed with 2% paraformoldehydefor 10 min at room temperature (this surface fixation doesn'tpermeabilize cells). Anti-TSG101 antibodies were incubated with bothcell lines for 20 min and detected with a fluorescent labeled secondaryantibody. The immuno-stained cells were analyzed via FACS. Top panel:H9ΔBg1 cells, with 85% cells stained positive for surface TSG101; bottompanel: H9 control cells, with less than 0.1% cells stained positive forsurface TSG101.

FIG. 6: Inhibition of HIV-1 production by anti-TSG101 antibodies. Lane 1and 5, mock transfection; Lane 2 and 6, pNL4-3 and control antibody(rabbit IgG); Lane 3 and 7, pNL4-3 and anti-TSG101 antibody “B”; Lane 4and 8, pNL4-3 and anti-TSG101 antibody “E”.

FIG. 7: Antibody Inhibition of HIV Release from H9ΔBg1 cells. HIVproducing H9ΔBg1 cells were incubated with anti-TSG101 antibody “E” atdifferent concentrations, 48 hours later, viral supernatants werecollected and assayed by HIV p24 ELISA kit. Averages of threeindependent experiments (each with triplicates) were shown. Significantantibody inhibition (*P<0.05) viral release was observed at 80 ug/ml.

FIG. 8: Antibody Inhibition of HIV Infectivity. HIV producing Jurkatcells (infected with Wild-type HIV-1) were incubated with anti-TSG101antibody “E” at 40 ug/ml, 48 hours later, viral supernatants werecollected and used for infection of MAGI cells, assayed by HIV p24 ELISAkit. Averages of three independent experiments (each with triplicates)were shown. Significant antibody inhibition of viral release wasobserved at 40 ug/ml.

FIGS. 9A-B: Release of Ebola GP and VP40 into culture supernatants. 9A293T cells were transfected with the indicated plasmids, supernatantswere cleared from floating and particulate material were pelletedthrough 20% sucrose by ultracentrifugation. The individual proteins weredetected in the cell lysates and in the particulate material fromsupernatant by immunoblotting (IB). 9B Supernatants from cellstransfected with Ebola VP40 alone or GP+vp40 WERE IMMUNPRECIPITATED WITHANTI-gp MaB AND ANALYZED BY IMMUNOBLOTTING. Lower panels shows theexpression of VP40 in total cell lysates. IgH: immunoglobulin heavychain from the antibody used for immunoprecipitation.

FIGS. 10A-B: Electron microscopic analysis of virus like particlesgenerated by EBOV GP and VP40. Particles obtained by ultracentrifugationof the supernatants of 293T cells transfected with Ebola GP+VP40 werenegatively stained with uranyl-acetate to reveal the ultrastructure(10A), or stained with anti-Ebo-GP mAb followed by Immunogold rabbitanti mouse Ab (10B), and analyzed by electron microscopy.

FIG. 11: Association of VP40 and TSG101 in 293T cells. Cells weretransfected with Myc tagged TSG101 full length (FL) or the indicatedtruncations along with VP40. After 48 h cells were lysed and subjectedto immunoprecipitation with anti Mye.

FIG. 12 shows Far-Western analysis of association between Ebola VP40 andTSG101 UEV domain.

FIG. 13 shows SPR analysis of Ebola VP40 interaction with TSG101.

FIGS. 14A-B: Association of TSG101 with Ebola VLPs and inactivated Ebolavirus. 14A 293 cells were transfected with the indicated plasmids,supernatants were immunoprecipitated with anti-GP mAb and analyzed byimmunoblotting with the antibodies indicated on the right. Lower threepanels shows the expression of the transfected proteins in total celllysates. 14B: 5 μg inactivated Ebola virus (iEBOV) were subjected toSDS-PAGE and Western blot analysis with rabbit anti-TSG101 antibody. Themolecular weight markers and position of TSG101 are indicated.

FIG. 15 shows results of inhibition of Ebola virus release in Hela cellsby anti TSG101 antibodies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of using antibodies that bind aTSG101 protein to inhibit or reduce viral production. The presentinvention also provides methods of using TSG101 antibodies for treatingviral infections. The present invention also provides methods andcompositions for treating viral infection by targeting TSG101 on thesurface of infected cells, e.g., delivering therapeutic and/ordiagnostic agents, to such infected cells.

The rational design of therapeutics requires an improved understandingof HIV pathogenesis. Recent studies show that a host protein, TSG101,plays a critical role in the pathogenesis of various viruses such as HIVand Ebola viruses. In particular, TSG101 participates in the process bywhich viral particles escape, or bud, from infected cells, and thereforerepresents a novel target or anti-viral drug discovery. Underlying theassembly and release of enveloped RNA viruses from infected cells is atight coordination between viral and host proteins (Perez et al., 2001,Immunity 15(5): 687-90; Freed, 2002, J. Virol. 76(10): 4679-87;Pornillos et al., 2002, Trends Cell Biol. 12(12): 569-79). While many ofthe protein: protein and protein: membrane interactions that govern thefinal stages of infection have yet to be identified, the cellular TSG101protein has emerged as a critical player (Garrus et al., 2001, Cell107:55-65; Carter 2002; Pornillos et al., 2002, Trends Cell Biol. 12(12): 569-79; Pornillios et al., 2002, Natl. Struct. Biol. 9, 812-7).Genetic, biochemical and microscopic analyses have shown that TSG101interacts with multiple enveloped RNA viruses—including members of theretrovirus, rhabdovirus and filovirus families.

Despite their considerable evolutionary divergence, many enveloped RNAviruses employ similar strategies to complete the final stages ofinfection (Martin-Serrano et al., 2001, Nature Medicine 7:1313-19;Freed, 2002, J. Virol. 76(10): 4679-87). Of particular importance toHuman Immunodeficiency Virus Type 1 (HIV-1), Vesicular Stomatitis Virus(VSV), Ebola Virus (EVOV), Marburg Virus (MARV) nad others is the Lateor L domain, a sequence motif that uniquely appropriates cellularpathways to drive viral particle assembly and budding. Three sequencemotifs with L-domain activity have been characterized: PPxY, YxxL andPTAP (where “x” denotes any amino acid)(SEQ ID NOS.: 5-7). HIV-1 buddingrequires the PTAP motif, found at the amino terminus of the p6Gagprotein. Rhabdoviruses, as typified by VSV, utilize the PPxY motif SEQID NO.: 5) within the Matrix (M) protein. The L-domains of multipleviral families recruit TSG101, a cellular protein critical to endosomalmembrane sorting (VerPlank et al., 2001, Proc. Natl. Acad. Sci. USA98:7724-7729; Pornillos et al., 2002, Natl Struct. Biol. 9, 812-7).Initially identified by a random knockout screen in mammalian cells,TSG101 is a 43KDa multifunctional protein involved in membranetrafficking, cell cycle control, microtubule assembly and proteindegradation (Li et al., 1996, Cell 85, 319-29; bishop et al., 2001, J.Biol. Chem. 276: 11735-42; Katzmann et al., 2001, Cell 106, 145-55; Liet al., 2001, Proc. Natl. Acad. Sci. USA 98, 1619-24. The C-terminus ofTSG101 possesses a coiled-coil domain and a domain that auto-regulatesits cellular levels; whereas the TSG101 amino-terminus which interactswith multiple viral L-domains via a binding pocket that structurally andfunctionally resembles WW and SH3 domains bears significant homology toUbiquitin Conjugating (UBC) E2 enzymes (Pornillos et al., 2002, Nat.Struct. Biol. 9, 812-7). Although the UBC-like domain of TSG101 stronglybinds ubiquitin, a 76 amino acid protein central to regulating proteinturnover and sorting, it lacks the catalytic cysteine residue involvedin ubiquitination of target proteins (Hicke, 2001, Cell 106(5): 527-30).

In eukaryotic cells, TSG101 is a component of ERCRT1 (endosomal sortingcomplex required for transport), a ˜350 kDa cytoplasmic complex thatalso includes Vps28 and Vps37 (Katzmann et al., 2001, Cell 106, 145-55;bishop et al., 2002, J. Cell Biol. 157, 91-101). The interaction amongthese three proteins, and their respective roles in membrane traffickingare currently under investigation. The TSG101 yeast homologue, Vps23,was identified by its functional complementation of protein sortingdefects (Babst et al., 2000, Traffic 1, 248-58). Fibroblasts withreduced TSG101 levels and yeast Vps23 null mutants both display defectsin the endosomal/MVB pathway. For instance, receptors that wouldnormally enter the MVB system for lysosomal degradation are insteadrecycled to the surface, leading to profound disturbances in cellsignaling. Based on their recent experimental analysis, Katzmann et al.have suggested that TSG101/Vps23 binds ubiquitinated proteins at thesurface of early endosomes, and facilitates their entry into MVBvesicles (Katzmann et al., 2001, Cell 106, 145-55).

In addition to TSG101, cellular proteins with WW-domains have been shownto interact with the L-domain sequence motifs of enveloped RNA viruses(Harty, et al., 2000, Proc. Nat'l. Acad. Sci. U.S.A. 97 (25): 13871-6;Kikonyogo et al., 2001, Proc. Nat'l. Acad. Sci. U.S.A. 98(20):11199-204). For example, Far-Western binding assays have demonstrated aspecific interaction with the WW-domains of the mammalian ubiquitinligase, Nedd4, and its yeast homolog Rsp5, with the VP40 L domain ofEBOV (Harty, et al., 2000, Proc. Nat'l. Acad. Sci. U.S.A.97(25):13871-6; Kikonyogo et al., 2001, Proc. Nat'l. Acad. Sci. U.S.A.98(20): 11199-204). Indeed, the data thus far point to an important rolefor ubiquitin in viral budding (Patnaik et al., 2000, Proc. Nat'l. Acad.Sci. U.S.A. 97, 13069-74; Carter, 2002, Trends Microbiol 10(5): 203-5;Myers et al., 2002, J. Virol 76(22): 11226-35). There may also be aconstitutive interaction between Nedd4 and TSG101. It has been suggestedthat HIV-1 may exploit Nedd4 and TSG101 to escape from infected cells ina manner wholly unrelated to the endosomal/MVB pathway. Nevertheless,TSG101 is widely regarded as a key host factor appropriated by virusesto drive viral release. The proposed TSG101/MVB link is based, in part,on the biophysical process of MVB formation, which is known to includethe invagination of the endosomal lipid bilayer away from the cytoplasmand towards the lumen (Patnaik et al., 2000, Proc. Nat'l Acad. Sci.U.S.A. 97, 13069-74; Jasenosky et al., 2001, J. Virol. 75(11): 5205-14).Enveloped RNA viruses face similar topological parameters: followingviral assembly on the inner leaflet of the membrane, the bilayer mustevaginate towards the extracellular milieu—again away from thecytoplasm. Devoid of any catalytic ability to split an otherwisethermodynamically stable bilayer, viruses apparently recruit endosomalmembrane factors for assistance. The TSG101: L domain interaction maythus provide a vital nexus between nascent virions and the endosomalmachinery that drives membrane fission and budding. As discussed above,TSG101, a constituent of ESCRT-1, sorts ubiquitinated proteins forinclusion in the MVB pathway. But this sorting may be subverted in cellsinfected with HIV and related enveloped RNA viruses. That is, ratherthan directing ubiquitinated proteins into the MVB pathway, TSG101 andits endosomal counterparts may direct the plasma membrane and itsassociated viral particles to evaginate, forming enveloped vesicles thatpinch off from the plasma membrane.

The molecular determinants that drive virion assembly and release arestill an area of active research, through some general conclusions haveemerged. First, the recruitment of TSG101 to the plasma membrane duringvirion maturation is absolutely required. The data supporting a centralTSG101 role are compelling: (i) overexpression of the TSG101 UBC domainstrans-dominantly disrupts VLP formation in HIV-1 Gag expressing cells(Demirov et al., 2002, Proc. Nat'l. Acad. Sci. U.S.A. 99:955-960); (ii)ablating TSG101 expression via RNA interference impairs HIV-1 budding(Garrus et al., 2001, Cell 107:55-65) and (iii) in both of theseinstances, electron microscopic analysis demonstrated viral particlestethered to the plasma membrane via membranous stalks, structurallysimilar to those found in cells expressing L-domain defective viruses.As shown by Martin-Serrano et al., L-domain point mutations thatpreclude TSG101 binding with the filoviral VP40 or HIV-1 p6Gag, markedlyreduce viral particle release from human cells, an effect that coincideswith the failure of TSG101 to colocalize with the viral proteins at thelipid bilayer (Martin-Serrano et al., 2001, Nature Medicine 7:1313-19).Related experiments demonstrated that the EVOV L-domain was able tosubstitute for the p6Gag L-domain, with no discernible effects on VLPrelease, underscoring the conserved nature of the enveloped RNA viralbudding mechanisms. Significantly, HIV-1 L-domain is dispensable onceTSG101 is directed fused to HIV-1 Gag. Therefore the primaryresponsibility of the L-domain is to recruit TSG101 to the plasmamembrane This interaction between TSG101 and viral L domains representsa novel target for the prevention and treatment of HIV, EBO V and MARVinfections (Luban, 2001, Nat. Med 7(12): 1278-80; Senior, 2001, DrugDiscov. Today 6(23): 1184-1186).

The inventor has discovered that anti-TSG101 antibodies can be used forinhibiting or reducing viral infections.

5.1 Anti-TSG101 Antibodies

The invention encompasses the use of an antibody that contains a bindingsite which specifically binds a TSG101 protein for inhibiting orreducing viral infection. Such anti-TSG101 antibodies can therefore beused as broad spectrum anti-viral agents. The term “antibody” as usedherein refers to immunoglobulin molecules. In one embodiment, theantibody binds a C-terminal region of a TSG101 protein. In a preferredembodiment, the antibody binds an epitope comprised in the amino acidregion QLRALMQKARKTAGLSDLY (SEQ ID NO:3). In another embodiment, theantibody binds an N-terminal region of a TSG101 protein. In a preferredembodiment, the antibody binds an epitope comprised in the amino acidregion VRETVNVITLYKDLKPVL (SEQ ID NO:2).

As used herein, “epitope” refers to an antigenic determinant, i.e., aregion of a molecule that provokes an immunological response in a hostor is bound by an antibody. This region can but need not compriseconsecutive amino acids. The term epitope is also known in the art as“antigenic determinant.” An epitope may comprise as few as three aminoacids in a spatial conformation which is unique to the immune system ofthe host. Generally, an epitope consists of at least five such aminoacids, and more usually consists of at least 8-10 such amino acids.Methods for determining the spatial conformation of such amino acids areknown in the art.

The invention also envisions the use of antibody fragments that containa binding site which specifically binds a TSG101 protein. Examples ofimmunologically active fragments of immunoglobulin molecules includeF(ab) and F(ab′)2 fragments which can be generated by treating theantibody with an enzyme such as pepsin or papain. Examples of methods ofgenerating and expressing immunologically active fragments of antibodiescan be found in U.S. Pat. No. 5,648,237 which is incorporated herein byreference in its entirety.

The immunoglobulin molecules are encoded by genes which include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regions, aswell as a myriad of immunoglobulin variable regions. Light chains areclassified as either kappa or lambda. Light chains comprise a variablelight (V_(L)) and a constant light (C_(L)) domain. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively.Heavy chains comprise variable heavy (V_(H)), constant heavy 1 (CH1),hinge, constant heavy 2 (CH2), and constant heavy 3 (CH3) domains. TheIgG heavy chains are further sub-classified based on their sequencevariation, and the subclasses are designated IgG1, IgG2, IgG3 and IgG4.

Antibodies can be further broken down into two pairs of a light andheavy domain. The paired V_(L) and V_(H) domains each comprise a seriesof seven subdomains: framework region 1 (FR1), complementaritydetermining region 1 (CDR1), framework region 2 (FR2), complementaritydetermining region 2 (CDR2), framework region 3 (FR3), complementaritydetermining region 3 (CDR3), framework region 4 (FR4) which constitutethe antibody-antigen recognition domain.

A chimeric antibody may be made by splicing the genes from a monoclonalantibody of appropriate antigen specificity together with genes from asecond human antibody of appropriate biologic activity. Moreparticularly, the chimeric antibody may be made by splicing the genesencoding the variable regions of an antibody together with the constantregion genes from a second antibody molecule. This method is used ingenerating a humanized monoclonal antibody wherein the complementaritydetermining regions are mouse, and the framework regions are humanthereby decreasing the likelihood of an immune response in humanpatients treated with the antibody (U.S. Pat. Nos. 4,816,567, 4,816,397,5,693,762; 5,585,089; 5,565,332 and 5,821,337 which are incorporatedherein by reference in their entirety).

An antibody suitable for use in the present invention may be obtainedfrom natural sources or produced by hybridoma, recombinant or chemicalsynthetic methods, including modification of constant region functionsby genetic engineering techniques (U.S. Pat. No. 5,624,821). Theantibody of the present invention may be of any isotype, but ispreferably human IgG1.

Antibodies exist for example, as intact immunoglobulins or can becleaved into a number of well-characterized fragments produced bydigestion with various peptidases, such as papain or pepsin. Pepsindigests an antibody below the disulfide linkages in the hinge region toproduce a F(ab)′2 fragment of the antibody which is a dimer of the Fabcomposed of a light chain joined to a VH—CH1 by a disulfide bond. TheF(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the F(ab)′2 dimer to aFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region. See Paul, ed., 1993, Fundamental Immunology, Third Edition(New York: Raven Press), for a detailed description of epitopes,antibodies and antibody fragments. One of skill in the art willrecognize that such Fab′ fragments may be synthesized de novo eitherchemically or using recombinant DNA technology. Thus, as used herein,the term antibody fragments includes antibody fragments produced by themodification of whole antibodies or those synthesized de novo.

As used herein, an antibody can also be a single-chain antibody (scFv),which generally comprises a fusion polypeptide consisting of a variabledomain of a light chain fused via a polypeptide linker to the variabledomain of a heavy chain.

The invention also encompasses the use of a polyclonal population ofanti-TSG101 antibodies for inhibiting or reducing viral infection. Asused herein, a polyclonal population of anti-TSG101 antibodies of thepresent invention refers to a population of anti-TSG101 antibodies,which comprises a plurality of different anti-TSG101 antibodies eachhaving a different binding specificity. In one embodiment, thepopulation of anti-TSG101 antibodies comprises antibodies that bind aC-terminal region of a TSG101 protein. In a preferred embodiment, thepopulation of anti-TSG101 antibodies comprises antibodies that bind oneor more epitopes comprised in the amino acid region QLRALMQKARKTAGLSDLY(SEQ ID NO:3). In another embodiment, the population of anti-TSG101antibodies comprises antibodies that bind an N-terminal region of aTSG101 protein. In a specific embodiment, the population of anti-TSG101antibodies comprises antibodies that bind one or more epitopes comprisedin the amino acid region VRETVNVITLYKDLKPVL (SEQ ID NO:2).

Preferably, the plurality of anti-TSG101 antibodies of the polyclonalpopulation includes specificities for different epitopes of TSG101protein. In preferred embodiments, at least 90%, 75%, 50%, 20%, 10%, 5%,or 1% of anti-TSG101 antibodies in the polyclonal population target thedesired epitopes. In other preferred embodiments, the proportion of anysingle anti-TSG101 antibody in the polyclonal population does not exceed90%, 50%, or 10% of the population. The polyclonal population comprisesat least 2 different anti-TSG101 antibodies with differentspecificities. More preferably, the polyclonal population comprises atleast 10 different anti-TSG101 antibodies. Most preferably, thepolyclonal population comprises at least 100 different anti-TSG101antibodies with different specificities.

5.2 Production of Anti-TSG101 Antibodies

TSG101 protein or a fragment thereof can be used to raise antibodieswhich bind TSG101 protein. Such antibodies include but are not limitedto polyclonal, monoclonal, chimeric, single chain, Fab fragments, and anFab expression library. In a preferred embodiment, anti C-terminalTSG101 antibodies are raised using an appropriate C-terminal fragment ofa TSG101 protein. Such antibodies are useful in inhibiting viralproduction.

5.2.1 Production of Monoclonal Anti-TSG101 Antibodies

Antibodies can be prepared by immunizing a suitable subject with aTSG101 protein or a fragment thereof as an immunogen. The antibody titerin the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized polypeptide. If desired, the antibody molecules can beisolated from the mammal (e.g., from the blood) and further purified bywell-known techniques, such as protein A chromatography to obtain theIgG fraction. In one embodiment, an anti-N terminal TSG101 antibody(also referred to as anti-TSG101 antibody “C”) is raised using anN-terminal fragment of the human TSG101 protein: VRETVNVITLYKDLKPVL (SEQID NO:2). In another embodiment, an anti-C terminal TSG101 antibody(also referred to as anti-TSG101 antibody “E”) is raised using aC-terminal fragment of the human TSG101 protein: QLRALMQKARKTAGLSDLY(SEQ ID NO:3).

At an appropriate time after immunization, e.g., when the specificantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975, Nature 256:495-497), the human B cellhybridoma technique by Kozbor et al. (1983, Immunol. Today 4:72), theEBV-hybridoma technique by Cole et al. (1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing hybridomas is well known (see Current Protocolsin Immunology, 1994, John Wiley & Sons, Inc., New York, N.Y.). Hybridomacells producing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindthe polypeptide of interest, e.g., using a standard ELISA assay.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies. For example, the monoclonal antibodiesmay be made using the hybridoma method first described by Kohler et al.,1975, Nature, 256:495, or may be made by recombinant DNA methods (U.S.Pat. No. 4,816,567). The term “monoclonal antibody” as used herein alsoindicates that the antibody is an immunoglobulin.

In the hybridoma method of generating monoclonal antibodies, a mouse orother appropriate host animal, such as a hamster, is immunized ashereinabove described to elicit lymphocytes that produce or are capableof producing antibodies that will specifically bind to the protein usedfor immunization (see, e.g., U.S. Pat. No. 5,914,112, which isincorporated herein by reference in its entirety).

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA.

Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor,1984, J. Immunol., 133:3001; Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)). Culture medium in which hybridoma cells are growing isassayed for production of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by hybridoma cells is determined by immunoprecipitation or byan in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immuno-absorbent assay (ELISA). The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson et al., 1980, Anal. Biochem., 107:220.

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103,Academic Press, 1986). Suitable culture media for this purpose include,for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cellsmay be grown in vivo as ascites tumors in an animal. The monoclonalantibodies secreted by the subclones are suitably separated from theculture medium, ascites fluid, or serum by conventional immunoglobulinpurification procedures such as, for example, protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody directed against a TSG101 protein or a fragmentthereof can be identified and isolated by screening a recombinantcombinatorial immunoglobulin library (e.g., an antibody phage displaylibrary) with the TSG101 protein or the fragment. Kits for generatingand screening phage display libraries are commercially available (e.g.,Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene antigen SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. Nos. 5,223,409 and 5,514,548; PCT PublicationNo. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO92/09690; PCT Publication No. WO 90/02809; Fuchs et al., 1991,Bio/Technology 9:1370-1372; Hay et al., 1992, Hum. Antibod. Hybridomas3:81-85; Huse et al., 1989, Science 246:1275-1281; Griffiths et al.,1993, EMBO J. 12:725-734.

In addition, techniques developed for the production of “chimericantibodies” (Morrison, et al., 1984, Proc. Natl. Acad. Sci., 81,6851-6855; Neuberger, et al., 1984, Nature 312, 604-608; Takeda, et al.,1985, Nature, 314, 452-454) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss etal., U.S. Pat. No. 4,816,397, which are incorporated herein by referencein their entirety.)

Humanized antibodies are antibody molecules from non-human specieshaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework region from a human immunoglobulinmolecule. (see e.g., U.S. Pat. No. 5,585,089, which is incorporatedherein by reference in its entirety.) Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCT PublicationNo. WO 87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. Nos. 4,816,567 and 5,225,539;European Patent Application 125,023; Better et al., 1988, Science240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al.,1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987,Canc. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; Shaw etal., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison 1985, Science229:1202-1207; Oi et al., 1986, Bio/Techniques 4:214; Jones et al.,1986, Nature 321:552-525; Verhoeyan et al., 1988, Science 239:1534; andBeidler et al., 1988, J. Immunol. 141:4053-4060.

Complementarity determining region (CDR) grafting is another method ofhumanizing antibodies. It involves reshaping murine antibodies in orderto transfer full antigen specificity and binding affinity to a humanframework (Winter et al. U.S. Pat. No. 5,225,539). CDR-graftedantibodies have been successfully constructed against various antigens,for example, antibodies against IL-2 receptor as described in Queen etal., 1989 (Proc. Natl. Acad. Sci. USA 86:10029); antibodies against cellsurface receptors-CAMPATH as described in Riechmann et al. (1988,Nature, 332:323; antibodies against hepatitis B in Cole et al. (1991,Proc. Natl. Acad. Sci. USA 88:2869); as well as against viralantigens-respiratory syncitial virus in Tempest et al. (1991,Bio-Technology 9:267). CDR-grafted antibodies are generated in which theCDRs of the murine monoclonal antibody are grafted into a humanantibody. Following grafting, most antibodies benefit from additionalamino acid changes in the framework region to maintain affinity,presumably because framework residues are necessary to maintain CDRconformation, and some framework residues have been demonstrated to bepart of the antigen binding site. However, in order to preserve theframework region so as not to introduce any antigenic site, the sequenceis compared with established germline sequences followed by computermodeling.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chain genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a TSG101 protein.

Monoclonal antibodies directed against a TSG101 protein can be obtainedusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA and IgE antibodies. For an overview ofthis technology for producing human antibodies, see Lonberg and Huszar(1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see e.g., U.S.Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825;U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. In addition,companies such as Abgenix, Inc. (Freemont, Calif., see, for example,U.S. Pat. No. 5,985,615) and Medarex, Inc. (Princeton, N.J.), can beengaged to provide human antibodies directed against a TSG101 protein ora fragment thereof using technology similar to that described above.

Completely human antibodies which recognize and bind a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al., 1994, Bio/technology12:899-903).

A pre-existing anti-TSG101 antibody can be used to isolate additionalantigens of the pathogen by standard techniques, such as affinitychromatography or immunoprecipitation for use as immunogens. Moreover,such an antibody can be used to detect the protein (e.g., in a cellularlysate or cell supernatant) in order to evaluate the abundance andpattern of expression of TSG101 protein. Detection can be facilitated bycoupling the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude 1251, 1311, 35S or 3H.

5.2.2. Production of Polyclonal Anti-TSG101 Antibodies

The anti-TSG101 antibodies can be produced by immunization of a suitableanimal, such as but are not limited to mouse, rabbit, and horse.

An immunogenic preparation comprising a TSG101 protein or a fragmentthereof are used to prepare antibodies by immunizing a suitable subject(e.g., rabbit, goat, mouse or other mammal). An appropriate immunogenicpreparation can contain, for example, recombinantly expressed orchemically synthesized TSG101 peptide or polypeptide. The preparationcan further include an adjuvant, such as Freund's complete or incompleteadjuvant, or similar immunostimulatory agent.

A fragment of a TSG101 protein suitable for use as an immunogencomprises at least a portion of the TSG101 protein that is 8 aminoacids, more preferably 10 amino acids and more preferably still, 15amino acids long.

The invention also provides chimeric or fusion TSG101 polypeptides foruse as immunogens. As used herein, a “chimeric” or “fusion” TSG101polypeptides comprises all or part of a TSG101 polypeptide operablylinked to a heterologous polypeptide. Within the fusion TSG101polypeptide, the term “operably linked” is intended to indicate that theTSG101 polypeptide and the heterologous polypeptide are fused in-frameto each other. The heterologous polypeptide can be fused to theN-terminus or C-terminus of the TSG101 polypeptide.

One useful fusion TSG101 polypeptide is a GST fusion TSG101 polypeptidein which the TSG101 polypeptide is fused to the C-terminus of GSTsequences. Such fusion TSG101 polypeptides can facilitate thepurification of a recombinant TSG101 polypeptide.

In another embodiment, the fusion TSG101 polypeptide contains aheterologous signal sequence at its N-terminus so that the TSG101polypeptide can be secreted and purified to high homogeneity in order toproduce high affinity antibodies. For example, the native signalsequence of an immunogen can be removed and replaced with a signalsequence from another protein. For example, the gp67 secretory sequenceof the baculovirus envelope protein can be used as a heterologous signalsequence (Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, 1992). Other examples of eukaryotic heterologoussignal sequences include the secretory sequences of melittin and humanplacental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yetanother example, useful prokaryotic heterologous signal sequencesinclude the phoA secretory signal and the protein A secretory signal(Pharmacia Biotech; Piscataway, N.J.).

In yet another embodiment, the fusion TSG101 polypeptide is animmunoglobulin fusion protein in which all or part of a TSG101polypetide is fused to sequences derived from a member of theimmunoglobulin protein family. The immunoglobulin fusion proteins can beused as immunogens to produce antibodies directed against the TSG101polypetide in a subject.

Chimeric and fusion TSG101 polypeptide can be produced by standardrecombinant DNA techniques. In one embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (e.g.,Ausubel et al., supra). Moreover, many expression vectors arecommercially available that already encode a fusion domain (e.g., a GSTpolypeptide). A nucleic acid encoding an immunogen can be cloned intosuch an expression vector such that the fusion domain is linked in-frameto the polypeptide.

The TSG101 immunogenic preparation is then used to immunize a suitableanimal. Preferably, the animal is a specialized transgenic animal thatcan secret human antibody. Non-limiting examples include transgenicmouse strains which can be used to produce a polyclonal population ofantibodies directed to a specific pathogen (Fishwild et al., 1996,Nature Biotechnology 14:845-851; Mendez et al., 1997, Nature Genetics15:146-156). In one embodiment of the invention, transgenic mice thatharbor the unrearranged human immunoglobulin genes are immunized withthe target immunogens. After a vigorous immune response against theimmunogenic preparation has been elicited in the mice, the blood of themice are collected and a purified preparation of human IgG molecules canbe produced from the plasma or serum. Any method known in the art can beused to obtain the purified preparation of human IgG molecules,including but is not limited to affinity column chromatography usinganti-human IgG antibodies bound to a suitable column matrix. Anti-humanIgG antibodies can be obtained from any sources known in the art, e.g.,from commercial sources such as Dako Corporation and ICN. Thepreparation of IgG molecules produced comprises a polyclonal populationof IgG molecules that bind to the immunogen or immunogens at differentdegree of affinity. Preferably, a substantial fraction of thepreparation are IgG molecules specific to the immunogen or immunogens.Although polyclonal preparations of IgG molecules are described, it isunderstood that polyclonal preparations comprising any one type or anycombination of different types of immunoglobulin molecules are alsoenvisioned and are intended to be within the scope of the presentinvention.

A population of antibodies directed to a TSG101 protein can be producedfrom a phage display library. Polyclonal antibodies can be obtained byaffinity screening of a phage display library having a sufficientlylarge and diverse population of specificities with a TSG101 protein or afragment thereof. Examples of methods and reagents particularly amenablefor use in generating and screening antibody display library can befound in, for example, U.S. Pat. Nos. 5,223,409 and 5,514,548; PCTPublication No. WO 92/18619; PCT Publication No. WO 91/17271; PCTPublication No. WO 92/20791; PCT Publication No. WO 92/15679; PCTPublication No. WO 93/01288; PCT Publication No. WO 92/01047; PCTPublication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs etal., 1991, Bio/Technology 9:1370-1372; Hay et al., 1992, Hum. Antibod.Hybridomas 3:81-85; Huse et al., 1989, Science 246:1275-1281; Griffithset al., 1993, EMBO J. 12:725-734. A phage display library permitsselection of desired antibody or antibodies from a very large populationof specificities. An additional advantage of a phage display library isthat the nucleic acids encoding the selected antibodies can be obtainedconveniently, thereby facilitating subsequent construction of expressionvectors.

In other preferred embodiments, the population of antibodies directed toa TSG101 protein or a fragment thereof is produced by a method using thewhole collection of selected displayed antibodies without clonalisolation of individual members as described in U.S. Pat. No. 6,057,098,which is incorporated by reference herein in its entirety. Polyclonalantibodies are obtained by affinity screening of a phage display libraryhaving a sufficiently large repertoire of specificities with, e.g., anantigenic molecule having multiple epitopes, preferably after enrichmentof displayed library members that display multiple antibodies. Thenucleic acids encoding the selected display antibodies are excised andamplified using suitable PCR primers. The nucleic acids can be purifiedby gel electrophoresis such that the full length nucleic acids areisolated. Each of the nucleic acids is then inserted into a suitableexpression vector such that a population of expression vectors havingdifferent inserts is obtained. The population of expression vectors isthen expressed in a suitable host.

5.2.3 Identifying Anti-TSG101 Antibodies that Inhibit Viral Production

The invention provides a method for identifying anti-TSG101 antibodiesthat can be used to inhibit or reduce viral budding. In one embodiment,the invention provides a method for determining the effect ofanti-TSG101 antibodies on viral infections using a retroviral infectionassay. A murine leukemia virus (MLV) derived vector which contains an E.coli lacZ gene expressed from the long terminal repeat (LTR) promoter(pBMN-Z-I-Neo) is transfected into an amphotropic murine leukemiaretroviral packaging cell line derived from 293 cells (Phoenix A, ATCC).Retroviruses produced by the Phoenix A helper cells are collected andused to infect a mouse N2A cells (ATCC). Anti-TSG101 antibodies areadded to 293 helper cells 24 hours after the transfection of the MLVvector. The effectiveness of TSG101 antibodies on viral production isdetermined by the efficiency of viral supernatant to infect the targetcells (N2A). The infection of N2A cells is then determined by cellularstaining of β-galactosidase activity (positive cells are stained blue,shown as dark spots in FIG. 2).

Typically, phoenix A cells are seeded on poly-D-lysine coated 6-wellplate a day before transfection. Four microgram of pBMN-Z-I-Neo is thentransfected into each well in the presence of 12 ul of Lipofectamine2000 (Invitrogen). Twenty-four hours post-transfection, media arereplaced with 1 ml/well of fresh media containing trichostatin A (3 uM)and 5 or 10 ug of proper anti-TSG101 antibodies. 24 to 48 hours later,viral supernatants are collected, filtered with 0.2 um filters, and 1 mlof viral supernatant is mixed with 1 ml of fresh media containingpolybrene (10 ug/ml), and then is used to infect one well of N2a cells.48 hours post-infection, N2a cells are fixed and stained with X-Gal asdescribed in the β-Gal staining kit (Invitrogen). Results are documentedby digital images. Preferably, anti-TSG101 antibodies that reduce viralproduction by at least 10%, 20%, 50%, 70% or 90% are identified.

In the following exemplary experiments, the two anti-TSG101 antibodies“C” and “E” are tested for their effect on viral infection. Rabbit IgGis used as non-specific antibody control. More than 10 independentexperiments are performed, and representative results are shown in FIG.2. Phoenix helper cells without treatment of antibody (positive control)showed efficient production of retroviruses, and infection of N2A targetcells (left top panel); Rabbit IgG had no effect (left middle panel).The anti-TSG101 antibody “C” reduced viral production by about 20%-60%(left bottom panel). The anti-TSG101 antibody “E” reduced viralproduction by about 50-70% (right top panel). A mixture ofanti-C-terminal and anti-N-terminal antibodies give similar results asthe anti-C terminal antibody alone (Right middle panel). N2a cells thatare not infected by viruses only showed minimal background staining(right bottom panel).

In another embodiment, anti-TSG101 antibodies that can be used toinhibit or reduce viral budding are identified based on their binding tocell surface TSG101, e.g., in a human CD4+ human T cell line H9transfected with HIV (designated as H9ΔBg1). H9ΔBg1 cells are human CD4+T lymphocytes transfected with an envelop-defective HIV construct(deletion of a Bg1 II fragment of HIV genome). The stably transfectedH9ΔBg1 cells produce a non-infectious form of HIV due to the defectiveHIV envelop, hence cannot infect other H9ΔBg1 cells in the culture. Inone embodiment, the untransfected H9 cells are used as control.Anti-TSG101 antibodies that bind to H9ΔBg1 but not the untransfected H9cells are identified as the antibodies that can be used to inhibit orreduce viral budding.

In a preferred embodiment, binding of an anti-TSG101 antibody to cellsurface TSG101 in HIV producing cells (e.g., H9ΔBg1) and control H9cells is identified by Fluorescence Activated Cell Sorting (FACS). Inone embodiment, both H9ΔBg1 and H9 cells are fixed, incubated withanti-TSG101 antibodies, and then stained with a fluorescence labeledsecondary antibody. The immuno-stained cells are then analyzed by FACS.

In another embodiment, a HIV-1 viral production assay is used to furtherexamine the inhibitory effect of TSG101 on retroviral production. TheHIV-1 vector pNL4-3 is transfected into 293T cells. 24 hours aftertransfection, an anti-TSG101 antibody and, optionally, a non-specificcontrol antibody are added respectively into the cell cultures. Afteradditional 24 hours incubation, cell lysates are extracted, cell culturesupernatants are collected and HIV-1 virions are purified, e.g., bysucrose gradients. Both cell lysates and purified virions are analyzedby Western blot using, e.g., anti-HIV-1 antibodies such as anti-p55and/or anti-p24 antibodies. Anti-TSG101 antibody that exhibitsignificant inhibition of HIV-1 virion release (e.g., more than 40%,50%, 60%, 70%, or 80% inhibition by density tracing of the Westernblots) can be identified.

In still another embodiment, the effect of an anti-TSG101 antibody onHIV release is evaluated using a HIV release assay based on H9ΔBg1cells. HIV release from H9ΔBg1 cells can be directly measured by HIV p24ELISA of cell culture supernatant. In one embodiment, a plurality ofdifferent concentrations of an anti-TSG101 antibody is incubatedrespectively with H9ΔBg1 cells. In one embodiment, a control antibody(e.g., rabbit IgG at the same concentrations) is also incubatedrespectively with H9ΔBg1 cells. 48 hours after antibody addition,culture supernatants are collected for HIV p24 ELISA. Effect of theanti-TSG101 antibody for inhibition of viral release is then determinedby comparing data of the anti-TSG101 antibody with the data of thecorresponding control antibody.

In still another embodiment, the effect of an anti-TSG110 antibody onHIV infectivity following viral release is determined. In oneembodiment, HIV supernatants from Jurkat cells are used to infect MAGIcells in the presence of an anti-TSG101 antibody. Rabbit IgG can be usedas controls. The anti-TSG101 antibody's effect on HIV infectivity isdetermined by comparing with the control.

5.3 Uses of Anti-TSG101 Antibodies for Treatment of Viral Infections

TSG101 antibodies are effective in inhibiting viral production. Theinvention therefore provides a method of treating viral infections,including HIV infection, using TSG101 antibodies, e.g., anti-C-terminalTSG101 antibodies.

5.3.1 Viral Infections

Diseases or disorders that can be treated or prevented by the use of ananti-TSG101 antibody of the present invention include, but are notlimited to, those caused by a ritrovirus, rhabdovirus, or filovirus,hepatitis type A, hepatitis type B, hepatitis type C, influenza,varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplextype II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsachie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II), any picornaviridae, enteroviruses, caliciviridae, anyof the Norwalk group of viruses, togaviruses, alphaviruses,flaviviruses, such as Dengue virus, coronaviruses, rabies virus, Marburgviruses, Ebola viruses, parainfluenza virus, orthomyxoviruses,bunyaviruses, arenaviruses, reoviruses, rotaviruses, orbiviruses, humanT cell leukemia virus type I, human T cell leukemia virus type II,simian immunodeficiency virus, lentiviruses, polyomaviruses,parvoviruses, Epstein-Barr virus, human herpesvirus-6, cercopithecineherpes virus 1 (B virus), and poxviruses. Additional diseases ordisorders that can be treated or prevented by the use of an anti-TSG101antibody of the present invention include, but are not limited to, thosecaused by influenza virus, human respiratory syncytial virus,pseudorabies virus, pseudorabies virus II, swine rotavirus, swineparvovirus, bovine viral diarrhea virus, Newcastle disease virus h,swine flu virus, swine flu virus, foot and mouth disease virus, hogcolera virus, swine influenza virus, African swine fever virus,infectious bovine rhinotracheitis virus, infectious laryngotracheitisvirus, La Crosse virus, neonatal calf diarrhea virus, Venezuelan equineencephalomyelitis virus, punta toro virus, murine leukemia virus, mousemammary tumor virus, equine influenza virus or equine herpesvirus,bovine respiratory syncytial virus or bovine parainfluenza virus.

5.3.2 Methods of Using Anti-TSG101 Antibodies for Inhibiting ViralRelease

In one embodiment, the present invention provides methods of usinganti-TSG101 antibodies, preferably anti-C-terminal TSG101 antibodies, ininhibiting or reducing viral budding, such as HIV-1 budding, frominfected mammalian cells. In the methods of the invention, one or moreanti-TSG101 antibodies are allowed to contact an infected cell. Theanti-TSG101 antibodies binds to the TSG101 protein on the surface of theinfected cell. The binding of the anti-TSG101 antibodies inhibits orreduces the release, or budding, of viral particles from the cell.

In another embodiment, the present invention thus also provides methodsusing anti-TSG101 antibodies, preferably anti-C-terminal TSG101antibodies, for treating infection by an enveloped virus, e.g., HIV-1,in a mammal, e.g., a human. In the methods of the invention, one or moreanti-TSG101 antibodies can be administered to an mammal, e.g., a human,infected by the virus. After administration, the anti-TSG101 antibodiesbind to TSG101 protein on the surface of an infected cell and inhibitingviral budding from the infected cell.

In still another embodiment of the invention, the anti-TSG101antibodies, preferably anti-C-terminal TSG101 antibodies are used inconjunction with one or more other therapeutic anti-viral drugs. In suchcombined therapies, the anti-TSG101 antibodies can be administeredbefore, at the same time, or after the administration of the therapeuticdrugs. The time intervals between the administration of the anti-TSG101antibodies and the therapeutic drugs can be determined by routineexperiments that are familiar to one skilled in the art.

In still another embodiment, the present invention provides a method fortreatment of viral infection using an anti-TSG101 antibody, e.g., ananti-C-terminal TSG101 antibody, that belongs to an isotype that iscapable of mediating the lysis of infected cells to which theanti-TSG101 antibody is bound. In a preferred embodiment, theanti-TSG101 antibody belongs to an isotype that binds a growth factorreceptor and activates serum complement and/or mediates antibodydependent cellular cytotoxicity (ADCC) by activating effector cells,e.g., macrophages. In another preferred embodiment, the isotype is IgG1,IgG2a, IgG3 or IgM.

The dosage of the anti-TSG101 antibodies can be determined by routineexperiments that are familiar to one skilled in the art. The effects orbenefits of administration of the anti-TSG01 antibodies can be evaluatedby any methods known in the art.

5.3.3 Methods of Using Anti-TSG101 Antibodies for Delivering Therapeuticand/or Diagnostic Agents

The invention provides methods and compositions for using anti-TSG101antibodies for delivering therapeutic and/or diagnostic agents to viralinfected cells.

Infected cells can be targeted and killed using anti-TSG101antibody-drug conjugates. For example, an anti-TSG101 antibody may beconjugated to a therapeutic moiety such as a cytotoxin, e.g., acytostatic or cytocidal agent, or a radioactive metal ion. Antibody-drugconjugates can be prepared by method known in the art (see, e.g.,Immunoconjugates, Vogel, ed. 1987; Targeted Drugs, Goldberg, ed. 1983;Antibody Mediated Delivery Systems, Rodwell, ed. 1988). Therapeuticdrugs, such as but are not limited to, paclitaxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof, can be conjugated to anti-TSG101 antibodies of theinvention. Other therapeutic agents that can be conjugated toanti-TSG101 antibodies of the invention include, but are not limited to,antimetabolites, e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine; alkylating agents, e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin; anthracyclines, e.g., daunorubicin (daunomycin) anddoxorubicin; antibiotics, e.g., dactinomycin (actinomycin), bleomycin,mithramycin, anthramycin (AMC); and anti-mitotic agents, e.g.,vincristine and vinblastine. The therapeutic agents that can beconjugated to anti-TSG101 antibodies of the invention may also be aprotein or polypeptide possessing a desired biological activity. Suchproteins may include, for example, a toxin such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin.

The drug molecules can be linked to the anti-TSG101 antibody via alinker. Any suitable linker can be used for the preparation of suchconjugates. In some embodiments, the linker can be a linker that allowsthe drug molecules to be released from the conjugates in unmodified format the target site.

The antibodies can also be used diagnostically to, for example, monitorthe progression of a viral infection as part of a clinical testingprocedure to, e.g., determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions. See generally U.S. Pat. No. 4,741,900 for metalions which can be conjugated to antibodies for use as diagnosticsaccording to the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include fluorescent proteins, e.g., greenfluorescent protein (GFP), umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In, 117Lu, ⁹⁰Y or ⁹⁹Tc.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982); each of which is incorporated herein byreference.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference.

5.3.4. Detection of Viral Infected Cells

Antibodies or labeled antibodies directed against a TSG101 protein,e.g., an N-terminal region or a C-terminal region of a TSG101 protein,may also be used as diagnostics and prognostics of viral infection,e.g., by detecting the presence of TSG101 protein on cell surface. Suchdiagnostic methods, may also be used to detect abnormalities in thelevel of TSG101 gene expression, or abnormalities in the structureand/or temporal, tissue, cellular, or subcellular location of a TSG101protein.

The tissue or cell type to be analyzed may include those which areknown, or suspected, to be infected by a virus. The protein isolationmethods employed herein may, for example, be such as those described inHarlow and Lane (Harlow, E. and Lane, D., 1988, “Antibodies: ALaboratory Manual”, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.), which is incorporated herein by reference in itsentirety. The isolated cells can be derived from cell culture or from apatient. The analysis of cell taken from culture may be a necessary stepin the assessment of cells to be used as part of a cell-based genetherapy technique or, alternatively, to test the effect of compounds onthe expression of the TSG101 gene.

Preferred diagnostic methods for the detection of TSG101 fragments orconserved variants or peptide fragments thereof, may involve, forexample, immunoassays wherein the TSG101 fragments or conserved variantsor peptide fragments are detected by their interaction with ananti-TSG101 fragment-specific antibody.

For example, antibodies, or fragments of antibodies, such as thosedescribed above useful in the present invention may be used toquantitatively or qualitatively detect infected cells by the presence ofTSG101 fragments or conserved variants or peptide fragments thereof ontheir surfaces. This can be accomplished, for example, byimmunofluorescence techniques employing a fluorescently labeled antibody(see below, this Section) coupled with light microscopic, flowcytometric, or fluorimetric detection. Such techniques are especiallyuseful in viral infection where TSG101 fragments are recruited to thecell surface during the viral budding process.

The antibodies (or fragments thereof) useful in the present inventionmay, additionally, be employed histologically, as in immunofluorescenceor immunoelectron microscopy, for in situ detection of TSG101 fragmentsor conserved variants or peptide fragments thereof. In situ detectionmay be accomplished by removing a histological specimen from a patient,and applying thereto a labeled antibody of the present invention. Theantibody (or fragment) is preferably applied by overlaying the labeledantibody (or fragment) onto a biological sample. Through the use of sucha procedure, it is possible to determine not only the presence of theTSG101 fragment, or conserved variants or peptide fragments, but alsoits distribution in the examined tissue. Using the present invention,those of ordinary skill will readily perceive that any of a wide varietyof histological methods (such as staining procedures) can be modified inorder to achieve such in situ detection.

Immunoassays for TSG101 fragments or conserved variants or peptidefragments thereof will typically comprise incubating a sample, such as abiological fluid, a tissue extract, freshly harvested cells, or lysatesof cells which have been incubated in cell culture, in the presence of adetectably labeled antibody capable of identifying TSG101 fragments orconserved variants or peptide fragments thereof, and detecting the boundantibody by any of a number of techniques well-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled TSG101 proteinspecific antibody. The solid phase support may then be washed with thebuffer a second time to remove unbound antibody. The amount of boundlabel on solid support may then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tub, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-Tsg101 fragment antibody maybe determined according to well known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

One of the ways in which the Tsg101 gene peptide-specific antibody canbe detectably labeled is by linking the same to an enzyme and use in anenzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked ImmunosorbentAssay (ELISA)”, 1978, Diagnostic

Horizons 2:1-7, Microbiological Associates Quarterly Publication,Walkersville, Md.); Voller, A. et al., 1978, J. Clin. Pathol.31:507-520; Butler, J. E., 1981, Meth. Enzymol. 73:482-523; Maggio, E.(ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa,E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). Theenzyme which is bound to the antibody will react with an appropriatesubstrate, preferably a chromogenic substrate, in such a manner as toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. The detection can be accomplishedby colorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect TSG101 peptides through theuse of a radioimmunoassay (RIA) (see, for example, Weintraub, B.,Principles of Radioimmunoassays, Seventh Training Course on RadioligandAssay Techniques, The Endocrine Society, March, 1986, which isincorporated by reference herein). The radioactive isotope can bedetected by such means as the use of a gamma counter or a scintillationcounter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

5.3.5. Depletion of Viral Infected Cells In Vitro

The invention provides methods of depleting viral infected cells fromnon infected tissues and/or cells in vitro (or ex vivo). For example,the tissue obtained from a mammal for the in vitro depletion of viralinfected cells from non infected cells can be blood or serum or otherbody fluid. In particular, the invention provides for methods ofdepleting viral infected cells by killing them or by separating themfrom non infected cells. In one embodiment, anti-TSG101 antibodies arecombined, e.g., incubated, in vitro with tissues and/or cells obtainedfrom a mammal, e.g., a human.

In one embodiment, a column containing a TSG101 antibody, e.g., anantibody that binds the N-Terminal or C-terminal region of a TSG101protein, bound to a solid matrix is used to remove viral infected cellsfrom a biological sample, e.g., blood or serum or other body fluid.

The anti-TSG101 antibodies used in the in vitro depletion of viralinfected cells from tissues can be conjugated to detectable labels(e.g., various enzymes, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials) or therapeuticagents (e.g., cytostatic and cytocidal agents), which are disclosed insection 5.3.2.

Anti-TSG101 antibodies conjugated to detectable substances can beutilized to sort viral infected cells from non infected cells by methodsknown to those of skill in the art. In one embodiment, viral infectedcells are sorted using a fluorescence activated cell sorter (FACS).Fluorescence activated cell sorting (FACS) is a well-known method forseparating particles, including cells, based on the fluorescentproperties of the particles (Kamarch, 1987, Methods Enzymol,151:150-165). Laser excitation of fluorescent moieties in the individualparticles results in a small electrical charge allowing electromagneticseparation of positive and negative particles from a mixture.

In one embodiment, cells, e.g, blood cells, obtained a mammal, e.g. ahuman, are incubated with fluorescently labeled TSG101 specificantibodies for a time sufficient to allow the labeled antibodies to bindto the cells. In an alternative embodiment, such cells are incubatedwith TSG101 specific antibodies, the cells are washed, and the cells areincubated with a second labeled antibody that recognizes the TSG101specific antibodies. In accordance with these embodiments, the cells arewashed and processed through the cell sorter, allowing separation ofcells that bind both antibodies to be separated from hybrid cells thatdo not bind both antibodies. FACS sorted particles may be directlydeposited into individual wells of 96-well or 384-well plates tofacilitate separation.

In another embodiment, magnetic beads can be used to separate viralinfected cells from non infected cells. Viral infected cells may besorted using a magnetic activated cell sorting (MACS) technique, amethod for separating particles based on their ability to bind magneticbeads (0.5-100 nm diameter) (Dynal, 1995). A variety of usefulmodifications can be performed on the magnetic microspheres, includingcovalent addition of antibody which immunospecifically recognizesTSG101. A magnetic field is then applied, to physically manipulate theselected beads. The beads are then mixed with the cells to allowbinding. Cells are then passed through a magnetic field to separate outviral infected cells.

5.3.6. Dose of Anti-TSG101 Antibodies

The dose can be determined by a physician upon conducting routine tests.Prior to administration to humans, the efficacy is preferably shown inanimal models. Any animal model for an infectious disease known in theart can be used.

In general, for antibodies, the preferred dosage is 0.1 mg/kg to 100mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibodyis to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usuallyappropriate. Generally, partially human antibodies and fully humanantibodies have a longer half-life within the human body than otherantibodies. Accordingly, lower dosages and less frequent administrationare often possible. Modifications such as lipidation can be used tostabilize antibodies and to enhance uptake and tissue penetration (e.g.,into the brain). A method for lipidation of antibodies is described byCruikshank et al., 1997, J. Acquired Immune Deficiency Syndromes andHuman Retrovirology 14:193.

As defined herein, a therapeutically effective amount of anti-TSG101antibody (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kgbody weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of an anti-TSG101 antibody can include a singletreatment or, preferably, can include a series of treatments. In apreferred example, a subject is treated with an anti-TSG101 antibody inthe range of between about 0.1 to 20 mg/kg body weight, one time perweek for between about 1 to 10 weeks, preferably between 2 to 8 weeks,more preferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. It will also be appreciated that the effectivedosage of an anti-TSG101 antibody, used for treatment may increase ordecrease over the course of a particular treatment. Changes in dosagemay result and become apparent from the results of diagnostic assays asdescribed herein.

It is understood that appropriate doses of anti-TSG101 antibody agentsdepends upon a number of factors within the ken of the ordinarilyskilled physician, veterinarian, or researcher. The dose(s) of theanti-TSG101 antibody will vary, for example, depending upon theidentity, size, and condition of the subject or sample being treated,further depending upon the route by which the composition is to beadministered, if applicable, and the effect which the practitionerdesires the anti-TSG101 antibody to have upon an infectious agent.

5.3.7. Pharmaceutical Formulation and Administration

The anti-TSG101 antibodies of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise anti-TSG101 antibody and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe anti-TSG101 antibody, use thereof in the compositions iscontemplated. Supplementary anti-TSG101 antibodies can also beincorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Preferred routesof administration include subcutaneous and intravenous. Other examplesof routes of administration include parenteral, intradermal, transdermal(topical), and transmucosal. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELM (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat the viscosity is low and the anti-TSG101 antibody is injectable. Itmust be stable under the conditions of manufacture and storage and mustbe preserved against the contaminating action of microorganisms such asbacteria and fungi.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theanti-TSG101 antibody (e.g., one or more anti-TSG101 antibodies) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theanti-TSG101 antibody into a sterile vehicle which contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In one embodiment, the anti-TSG101 antibodies are prepared with carriersthat will protect the compound against rapid elimination from the body,such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811 which is incorporated herein by reference in its entirety.

It is advantageous to formulate parenteral compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of anti-TSG101 antibody calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the anti-TSG101 antibody and the particulartherapeutic effect to be achieved, and the limitations inherent in theart of compounding such an anti-TSG101 antibody for the treatment ofindividuals.

The pharmaceutical compositions can be included in a kit, in acontainer, pack, or dispenser together with instructions foradministration.

5.4 TSG101 Vaccines and DNA Vaccines for Treatment and Prevention ofViral Infection

The invention provides fragments of a TSG101 protein which can be usedas vaccines to generate anti-TSG101 antibodies. The TSG101 proteinfragment or polypeptide can be prepared by standard method known in theart. In one embodiment, the invention provides a fragment of a TSG101protein not comprising the UEV domain of a TSG101 protein. In a specificembodiment, the invention provides a fragment of a human TSG101 protein,or its murine homolog, not comprising the UEV domain. In a preferredembodiment, the invention provides a fragment comprising the C-terminalregion of a TSG101 protein. In another embodiment, the inventionprovides a fragment of a TSG101 protein comprising the coiled-coildomain of a TSG101 protein. In still another embodiment, the inventionprovides a fragment of a TSG101 protein comprising C-terminal domain ofa TSG101 protein as described SEQ ID NO:3. The invention also providesany sequence that is at least 30%, 50%, 70%, 90%, or 95% homologous suchfragments of a TSG101 protein. In some embodiments of the invention, theTSG101 protein fragments or polypeptides are at least 5, 10, 20, 50, 100amino acids in length.

The invention also provides fragment of a TSG101 protein which isfunctionally equivalent to any TSG101 fragment described above. Such anequivalent TSG101 fragment may contain deletions, additions orsubstitutions of amino acid residues within the amino acid sequenceencoded by the TSG101 protein gene sequences encoding the TSG101 proteinbut which result in a silent change, thus producing a functionallyequivalent TSG101 protein fragment. Amino acid substitutions may be madeon the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. For example, nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine; polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;positively charged (basic) amino acids include arginine, lysine, andhistidine; and negatively charged (acidic) amino acids include asparticacid and glutamic acid. “Functionally equivalent”, as utilized herein,refers to a protein fragment capable of exhibiting a substantiallysimilar in vivo activity as the endogenous TSG101 protein fragment.

The TSG101 peptide fragments of the invention may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing the TSG101 polypeptides and peptides of theinvention by expressing nucleic acid containing TSG101 gene sequencesencoding the TSG101 polypeptide or peptide. Methods which are well knownto those skilled in the art can be used to construct expression vectorscontaining TSG101 polypeptide coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. See, for example, thetechniques described in Sambrook et al., 1989, supra, and Ausubel etal., 1989, supra. Alternatively, RNA capable of encoding TSG101polypeptide sequences may be chemically synthesized using, for example,synthesizers. See, for example, the techniques described in“Oligonucleotide Synthesis”, 1984, Gait, M. J. ed., IRL Press, Oxford,which is incorporated herein by reference in its entirety.

The TSG101 peptide can be used in combination with a suitable carrierand/or adjuvant, such as Freund's complete or incomplete adjuvant, or asimilar immunostimulatory agent. An oil/surfactant based adjuvantcomprising one or more surfactants combined with one or morenon-metabolizable mineral oil or metabolizable oil, such as theIncomplete Seppic Adjuvant (Seppic, Paris, France), may be used. AnIncomplete Seppic Adjuvant has comparable effect as Incomplete Freund'sAdjuvant for antibody production, but induces lower inflammatoryresponse.

The invention also provides portions of a tsg101 gene for use as DNA orRNA vaccine. The tsg101 gene fragments can also be used for producingany TSG101 protein fragment of the invention described above. In apreferred embodiment, the invention provides a fragment of a tsg101 genecomprising the nucleotide region encoding a fragment not comprising theUEV domain of a TSG101 protein. In a specific embodiment, the fragmentof a TSG101 gene is a fragment of a human tsg101 gene, or its murinehomolog. The invention also provides any sequence that is at least 30%,50%, 70%, 90%, or 95% homologous to such fragments of a tsg101 gene. Insome embodiments of the invention, the fragment of a tsg101 gene is atleast 20, 25, 40, 60, 80, 100, 500, 1000 bases in length. Such sequencesmay be useful for production of TSG101 peptides.

The invention also provides (a) DNA vectors that contain any of theforegoing tsg101 coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingtsg101 coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences; and (c) geneticallyengineered host cells that contain any of the foregoing TSG101 codingsequences operatively associated with a regulatory element that directsthe expression of the coding sequences in the host cell for use inproducing a TSG101 protein fragment of the invention. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the cytomegalovirushCMV immediate early gene, the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage A, the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinase,the promoters of acid phosphatase, and the promoters of the yeast.alpha.-mating factors.

In another embodiment, the present invention provides a naked DNA or RNAvaccine, and uses thereof. The tsg101 DNA fragment of the presentinvention described above can be administered as a vaccine to inhibitviral disease by eliciting anti-TSG101 antibodies of the invention. TheDNA can be converted to RNA for example by subcloning the DNA into atranscriptional vector, such as pGEM family of plasmid vectors, or undercontrol of a transcriptional. promoter of a virus such as vaccinia, andthe RNA used as a naked RNA vaccine. The naked DNA or RNA vaccine can beinjected alone, or combined with one or more DNA or RNA vaccinesdirected to the virus.

The naked DNA or RNA vaccine of the present invention can beadministered for example intermuscularly, or alternatively, can be usedin nose drops. The DNA or RNA fragment or a portion thereof can beinjected as naked DNA or RNA, as DNA or RNA encapsulated in liposomes,as DNA or RNA entrapped in proteoliposomes containing viral envelopereceptor proteins (Nicolau, C. et al. Proc. Natl. Acad. Sci. U.S.A.1983, 80, 1068; Kanoda, Y., et al. Science 1989, 243, 375; Mannino, R.J. et al. Biotechniques 1988, 6, 682). Alternatively, the DNA can beinjected along with a carrier. A carrier can be a protein or such as acytokine, for example interleukin 2, or a polylysine-glycoproteincarrier (Wu, G. Y. and Wu, C. H. J. Biol. Chem. 1988, 263, 14621), or anonreplicating vector, for example expression vectors containing eitherthe Rous sarcoma virus or cytomegalovirus promoters. Such carrierproteins and vectors and methods for using same are known to a person inthe art (See for example, Acsadi, G. et al. Nature 1991, 352, 815-818).In addition, the DNA or RNA could be coated onto tiny gold beads and thebeads introduced into the skin with, for example, a gene gun (Cohen, J.Science 1993, 259, 1691-1692; Ulmer, J. B. et al. Science 1993, 259,1745-1749).

The invention also provides methods for treating a viral infection,e.g., HIV infection, in an animal by gene therapy. A variety of genetherapy approaches may be used to introduce nucleic acid encoding afragment of the Tsg101 protein in vivo into cells so as to produceTsg101 antibodies.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow. For general reviews of the methods of gene therapy, see Goldspielet al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, New York; and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, New York.

In a preferred aspect, the therapeutic comprises a TSG101 nucleic acidthat is part of an expression vector that expresses a TSG101 or fragmentor chimeric protein thereof in a suitable host. In particular, such anucleic acid has a promoter operably linked to the TSG101 coding region,said promoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, a nucleic acidmolecule is used in which the TSG101 coding sequences and any otherdesired sequences are flanked by regions that promote homologousrecombination at a desired site in the genome, thus providing forintrachromosomal expression of the Tsg101 nucleic acid (see e.g., Kollerand Smithies, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:8932-8935; Zijlstraet al., 1989, Nature 342:435-438).

Delivery of the nucleic acid into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, or by administering it in linkage to a peptide which isknown to enter the nucleus, by administering it in linkage to a ligandsubject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J.Biol. Chem. 262:4429-4432) (which can be used to target cell typesspecifically expressing the receptors), etc. In another embodiment, anucleic acid-ligand complex can be formed in which the ligand comprisesa fusogenic viral peptide to disrupt endosomes, allowing the nucleicacid to avoid lysosomal degradation. In yet another embodiment, thenucleic acid can be targeted in vivo for cell specific uptake andexpression, by targeting a specific receptor (see, e.g., PCTPublications WO 92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635dated Dec. 23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992(Findeis et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.), WO93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, 1989,Proc. Natl. Acad. Sci. U.S.A. 86:8932-8935; Zijlstra et al., 1989,Nature 342:435-438).

In a specific embodiment, a viral vector that contains the TSG101nucleic acid is used. For example, a retroviral vector can be used (seeMiller et al., 1993, Meth. Enzymol. 217:581-599). These retroviralvectors have been modified to delete retroviral sequences that are notnecessary for packaging of the viral genome and integration into hostcell DNA. The Tsg101 nucleic acid to be used in gene therapy is clonedinto the vector, which facilitates delivery of the gene into a patient.More detail about retroviral vectors can be found in Boesen et al.,1994, Biotherapy 6:291-302, which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141;and Grossman and Wilson, 1993, Curr. Opin. Genet. and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson (1993,Current Opinion in Genetics and Development 3:499-503) present a reviewof adenovirus-based gene therapy. Bout et al. (1994, Human Gene Therapy5:3-10) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; andMastrangeli et al., 1993, J. Clin. Invest. 91:225-234.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In a preferred embodiment, epithelial cellsare injected, e.g., subcutaneously. In another embodiment, recombinantskin cells may be applied as a skin graft onto the patient. Recombinantblood cells (e.g., hematopoietic stem or progenitor cells) arepreferably administered intravenously. The amount of cells envisionedfor use depends on the desired effect, patient state, etc., and can bedetermined by one skilled person in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy, aTSG101 nucleic acid is introduced into the cells such that it isexpressible by the cells or their progeny, and the recombinant cells arethen administered in vivo for therapeutic effect. In a specificembodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells which can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment of the presentinvention. Such stem cells include but are not limited to hematopoieticstem cells (HSC), stem cells of epithelial tissues such as the skin andthe lining of the gut, embryonic heart muscle cells, liver stem cells(PCT Publication WO 94/08598), and neural stem cells (Stemple andAnderson, 1992, Cell 71:973-985).

Epithelial stem cells (ESCs) or keratinocytes can be obtained fromtissues such as the skin and the lining of the gut by known procedures(Rheinwald, 1980, Meth. Cell Bio. 21A:229). In stratified epithelialtissue such as the skin, renewal occurs by mitosis of stem cells withinthe germinal layer, the layer closest to the basal lamina. Stem cellswithin the lining of the gut provide for a rapid renewal rate of thistissue. ESCs or keratinocytes obtained from the skin or lining of thegut of a patient or donor can be grown in tissue culture (Rheinwald,1980, Meth. Cell Bio. 21A:229; Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771). If the ESCs are provided by a donor, a method forsuppression of host versus graft reactivity (e.g., irradiation, drug orantibody administration to promote moderate immunosuppression) can alsobe used.

With respect to hematopoietic stem cells (HSC), any technique whichprovides for the isolation, propagation, and maintenance in vitro of HSCcan be used in this embodiment of the invention. Techniques by whichthis may be accomplished include (a) the isolation and establishment ofHSC cultures from bone marrow cells isolated from the future host, or adonor, or (b) the use of previously established long-term HSC cultures,which may be allogeneic or xenogeneic. Non-autologous HSC are usedpreferably in conjunction with a method of suppressing transplantationimmune reactions of the future host/patient. In a particular embodimentof the present invention, human bone marrow cells can be obtained fromthe posterior iliac crest by needle aspiration (see e.g., Kodo et al.,1984, J. Clin. Invest. 73:1377-1384). In a preferred embodiment of thepresent invention, the HSCs can be made highly enriched or insubstantially pure form. This enrichment can be accomplished before,during, or after long-term culturing, and can be done by any techniquesknown in the art. Long-term cultures of bone marrow cells can beestablished and maintained by using, for example, modified Dexter cellculture techniques (Dexter et al., 1977, J. Cell Physiol. 91:335) orWitlock-Witte culture techniques (Witlock and Witte, 1982, Proc. Natl.Acad. Sci. U.S.A. 79:3608-3612).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Additional methods that can be adapted for use to deliver a nucleic acidencoding a Tsg101 fragment of the invention or functional derivativethereof are described below.

5.5 Kits

The invention also provides kits containing the anti-TSG101 antibodiesof the invention, or one or more TSG101 polypeptides which can be usedto raise anti-TSG101 antibodies, or one or more nucleic acids encodingpolypeptide anti-TSG101 antibodies of the invention, or cellstransformed with such nucleic acids, in one or more containers. Thenucleic acids can be integrated into the chromosome, or exist as vectors(e.g., plasmids, particularly plasmid expression vectors). Kitscontaining the pharmaceutical compositions of the invention are alsoprovided.

6. EXAMPLES

The following examples are presented by way of illustration of thepresent invention, and are not intended to limit the present inventionin any way.

6.1 Example 1 Preparation and Uses of Anti-TSG101 Antibodies

To determine the effect of anti-TSG101 antibodies on viral infections, aretroviral infection assay was developed. A murine leukemia virus (MLV)derived vector which contains an E. coli lacZ gene expressed from thelong terminal repeat (LTR) promoter (pBMN-Z-I-Neo) was transfected intoan amphotropic murine leukemia retroviral packaging cell line derivedfrom 293 cells (Phoenix A, ATCC). Retroviruses produced by the Phoenix Ahelper cells were collected and used to infect a mouse N2A cells (ATCC).Anti-TSG101 antibodies were added to 293 helper cells 24 hours after thetransfection of the MLV vector. The effectiveness of TSG101 antibodieson viral production was determined by the efficiency of viralsupernatant to infect the target cells (N2A). The infection of N2A cellswas then determined by cellular staining of β-galactosidase activity(positive cells were stained blue, showed as dark spots in FIG. 2).

Typically, phoenix A cells were seeded on poly-D-lysine coated 6-wellplate a day before transfection. Four microgram of pBMN-Z-I-Neo was thentransfected into each well in the presence of 12 ul of Lipofectamine2000 (Invitrogen). Twenty-four hours post-transfection, media werereplaced with 1 ml/well of fresh media containing trichostatin A (3 uM)and 5 or 10 ug of proper anti-TSG101 antibodies. 24 to 48 hours later,viral supernatants were collected, filtered with 0.2 um filters, and 1ml of viral supernatant was mixed with 1 ml of fresh media containingpolybrene (10 ug/ml), and then used to infect one well of N2a cells. 48hours post-infection, N2a cells were fixed and stained with X-Gal asdescribed in the β-Gal staining kit (Invitrogen). Results weredocumented by digital images.

In the following experiments, two anti-TSG101 antibodies were tested fortheir effect on viral infection, a rabbit antibody against N-terminalTSG101 protein, and a rabbit antibody against C-terminal TSG101 protein.The anti-N terminal TSG101 antibody was raised using a N-terminalfragment of the human TSG101 protein: VRETVNVITLYKDLKPVL (SEQ ID NO:2).The anti-C terminal TSG101 antibody was raised using a C-terminalfragment of the human TSG101 protein: QLRALMQKARKTAGLSDLY (SEQ ID NO:3).Rabbit IgG was used as non-specific antibody control. More than 10independent experiments were performed, and representative results areshown in FIG. 2. Phoenix helper cells without treatment of antibody(positive control) showed efficient production of retroviruses, andinfection of N2A target cells (left top panel); Rabbit IgG had no effect(left middle panel). The rabbit antibody against N-terminal TSG101showed about 20%-60% inhibition (left bottom panel). But the rabbitantibody against C-terminal TSG101 significantly inhibited theproduction of retroviruses, and infection of N2A target cells (50-70%inhibition, right top panel). A mixture of the anti-C terminal andanti-N terminal antibodies gave similar results as the anti-C terminalantibody alone (Right middle panel). N2a cells that were not infected byviruses only showed minimal background staining (right bottom panel).

Similar results were also obtained in HIV viral infection assays.

6.2 Example 2 TSG101 Localized on Cell Surface During Viral Budding

This example shows that domains of TSG101 are exposed on cell surfaceduring HIV release, and anti-TSG101 antibodies inhibited HIV release andinfection.

1. TSG101 Localization During Viral Release

To demonstrate TSG101 is actively involved viral release at plasmamembrane, an expression vector of GFP-TSG101 fusion protein wasconstructed and transfected into Phoenix cells (a retroviral helper cellline developed by Nolan et al of Stanford university) that was activelyproducing M-MuLV viruses. 24 hours after transfection, GFP-TSG101 fusionprotein traffic was observed under confocal microscope (Ultraview,Perkin-Elmer). FIGS. 3A-E show a time course of images of GFP-TSG101protein translocation from cytoplasm onto cell surface, and then“budding” out of the viral producing cells.

2. Cell Surface Localization of TSG101 During HIV Budding

To determine if TSG101 is also actively involved in HIV budding,anti-TSG101 antibodies were used to directly detect cell surface TSG101in a human CD4+ human T cell line H9 transfected with HIV (designated asH9ΔBg1), and the untransfected H9 cells were used as control. The tworabbit anti-TSG101 polyclonal antibodies, one against N-terminal(designated as anti-TSG101 “C”) and one against C-terminal TSG101(designated as anti-TSG101 “E”), were used for this study. Bothantibodies have been well characterized (Li et al., 2001, Proc Natl AcadSci USA 98(4): 1619-24). Both antibodies specifically detected cellsurface localization of TSG101 only in HIV producing H9ΔBg1 cells, andno cell surface TSG101 was detected in control H9 cells (FIG. 4).Interestingly, anti-TSG101 antibodies detected a “capping” like buddingstructure as observed with anti-HIV antibodies (Lee et al., 1999, J.Virol. 73:5654-62).

3. FACS Profile of Cell Surface Localization of TSG101 During HIVBudding

Cell surface localization of TSG101 in HIV producing cells (H9ΔBg1) andcontrol H9 cells was then examined by Fluorescence Activated Cell Sorter(FACS). Both H9ΔBg1 and H9 cells were fixed, stained with anti-TSG101antibodies, and detected with a fluorescence labeled secondary antibody.The immuno-stained cells were analyzed on FACS. More than sixindependent experiments showed that about 70-85% H9ΔBg1 cells werestained positive for surface TSG101, while less than about 0.1% H9control cells were stained positive for surface TSG101 (FIG. 5). Theseresults were further confirmed by direct examination of both H9ΔBg1 andH9 control cells under confocal microscope. The small population (lessthan 0.1%) of H9 control cells resulted from weak backgroundfluorescence signals associated with immunostaining procedure afterconfocal microscope analysis. The positive population of H9ΔBg1 cellsshowed bright fluorescence.

4. Anti-TSG101 Antibody Inhibition of HIV Production in Transfected 293Cells

A HIV-1 viral production assay was used to further examine theinhibitory effect of TSG101 on retroviral production. The HIV-1 vectorpNL4-3 was transfected into 293T cells. 24 hours after transfection, twoanti-TSG101 antibodies (10 ug/ml), anti-TSG101 antibody “E” andanti-TSG101 antibody “B”, and a non-specific control antibody (10 ug/ml)were added respectively into the cell cultures. Anti-TSG101 antibody “B”was raise against a murine TSG101 N-terminal fragment and binds poorlyto human TSG101 protein. Anti-TSG101 antibody “B” was used as a control.After an additional 24 hours incubation, cell lysates were extracted,cell culture supernatants were collected and HIV-1 virions were purifiedby sucrose gradients. Both cell lysates and purified virions wereanalyzed by Western blot using two anti-HIV-1 antibodies (anti-p55 andanti-p24). As shown in FIG. 6, anti-TSG101 antibody “E” treatment showedsignificant inhibition of HIV-1 virion release (more than 70% inhibitionby density tracing of the Western blots, Lane 8), while anti-TSG101antibody “B” (Lane 7) and the control antibody (Lane 6) did not showsignificant inhibition of HIV-1 release.

5. Antibody Inhibition of HIV Release from Human CD4+ T Lymphocytes(H9ΔBg1 Cells)

To specifically examine the effect of an antibody on HIV release, a HIVrelease assay based on H9ΔBg1 cells was developed. H9ΔBg1 cells arehuman CD4+ T lymphocytes transfected with an envelop-defective HIVconstruct (deletion of a Bg1 II fragment of HIV genome). The stablytransfected H9ΔBg1 cells produce a non-infectious form of HIV (due tothe defective HIV envelop, hence cannot infect other H9ΔBg1 cells in theculture), HIV release from H9ΔBg1 cells can be directly measured by HIVp24 ELISA of cell culture supernatant. Several concentrations of TSG101antibody “E” and control antibody (rabbit IgG at the sameconcentrations) were used to incubate with H9ΔBg1 cells. 48 hours afterantibody addition, culture supernatants were collected for HIV p24ELISA. Significant antibody inhibition of viral release was observed at80 ug/ml (FIG. 7).

6. Antibody Inhibition of HIV Infectivity

To determine if anti-TSG101 antibody has additional effect on HIVinfectivity following viral release, HIV supernatants from Jurkat cellswere used to infect MAGI cells in the presence of anti-TSG101 antibody“E” and rabbit IgG as controls (40 ug/ml). Anti-TSG101 antibody showedsignificant inhibition of HIV infectivity (FIG. 8), suggesting TSG101has a role in HIV maturation and/or infection of target cells followingviral release.

6.3 Example 3 Anti-TSG101 Antibodies Inhibit Release of Ebola Virus

This Example shows that TSG101 interacts with EBOV VP40, that TSG101 isincorporated into EBOV VLPs, and that anti-TSG101 antibody inhibits therelease of EBOV virus-like particles (VLPs).

The only members of the family Filoviridae, EBOV and MARV possess anegative-stranded, non-segmented 19 Kb RNA genome comprising 7 genes:nucleoprotein (NP), viral proteins VP35, VP40, glycoprotein (GP), VP30,VP24, and RNA polymerase (L), encoding for seven proteins in MARV andeight proteins in EBOV. Recent studies provide some insights in thecellular localization and role of VP40, a 326 amino acid matrix (M)protein (Jasenosky et al., 2001, J Virol 75(11): 5205-14; Kolesnikova etal., 2002, J Virol 76(4): 1825-38). In cells infected with either EBOVor MARV, the majority of VP40 is peripherally associated with thecytoplasmic face of the plasma membrane via hydrophobic interactions.Significantly, expression of EBOV and MARV VP40 in transfected cells isrequired for the production of virus-like particles (VLPs),non-infectious particles that have some morphological properties similarto authentic viruses. The ability of VP40 to direct its own release frominfected cells was mapped to a proline-rich sequence motif common toother enveloped RNA viruses (Harty et al., 2000, Proc Natl Acad Sci USA97(25): 13871-6).

1. Generation of Virus-Like Particles as a Surrogate Model for EbolaAssembly and Release

Several virus-like particles can be generated by mere expression ofviral matrix proteins (Johnson et al., 2000, Curr Opin Struct Biol 10,229-235). EBOV and MARV matrix proteins (VP40) have been shown tolocalize to both the plasma membrane and viral inclusion bodies(Kolesnikova et al., 2002, J Virol 76(4): 1825-38; Martin-Serrano et.al., 2001, Nature Medicine 7:1313-19), suggesting that VP40 may drivethe assembly and release of mature virions. However, attempts toefficiently generate VLPs by expression of VP40 alone have been largelyunfruitful, marked by inefficient release of amorphous VP40-containingmaterial (Bavari et al., 2002, J Exp Med 195, 593-602). In retroviruses,the raft localization of the assembly complex is regulated by theassociation of N-terminally acylated Gag proteins (Campbell, et al.,2001, J Clin Virol 22, 217-227), whereas raft targeting of filovirusproteins such as VP40 appear to be mainly regulated by the viralglycoprotein (GP) (Bavari et al., 2002, J Exp Med 195, 593-602).Therefore, it was hypothesized that generation of filovirus VLPs mayrequire coexpression of both GP and VP40. Whether GP and VP40 arereleased into culture supernatants was first examined. In cellsexpressing either GP or VP40 alone both proteins could be detected bothin cells and supernatants (FIG. 9A). Coexpression of both proteins,however, resulted in substantial increase in release from cells (FIG.9A). It was reasoned that if the released GP and VP40 are associated inparticles, VP40 must be co-immunoprecipitated with anti-GP mAb. As shownin FIG. 9B, VP40 was readily detected in anti GP-immunoprecipitates fromthe supernatants of cells transfected with both GP and VP40 of EBOV. NoVP40 was pulled down from the supernatant of cells expressing VP40alone, showing that the co-IP is specific.

2. Particles Formed by EBOV GP and VP40 Display the MorphologicalCharacteristics of Ebola Virus

The co-IP experiments demonstrated that GP and VP40 released intosupernatant are associated with each other in some form. To determinewhether these complexes represent virus-like particles (VLPs),particulate material from culture supernatants was purified by sucrosegradient ultracentrifugation (Bavari et al., 2002, J Exp Med 195,593-602) and analyzed using electron microscopy. Interestingly, most ofthe particles obtained from the supernatants of the cells cotransfectedwith GP and VP40 displayed a filamentous morphology strikingly similarto filoviruses (FIGS. 10A and B) (Geisbert, et al., 1995, Virus Res 39,129-150; Murphy et al., 1978, Ebola and Marburg virus morphology andtaxonomy, 1 edn (Amsterdam, Elsvier)). In contrast, the materialobtained from singly transfected cells only contained small quantitiesof membrane fragments, likely released during cell death. The VLPs havea diameter of 50-70 nm and are 1-2 μm in length (FIG. 10). This issimilar to the length range of Ebola virions found in cell culturesupernatants after in vitro infection (Geisbert, et al., 1995, Virus Res39, 129-150). The smaller diameter of VLPs (as compared to 80 nm forEBOV) may be due to the lack of ribonucleoprotein complex. All types ofmorphologies described for filoviruses such as branched, rod-, U- and6-shaped forms (Feldmann et al., 1996, Adv Virus Res 47, 1-52; Geisbert,et al., 1995, Virus Res 39, 129-150) among these particles were observed(FIG. 10). In addition, the VLPs were coated with 5-10 nm surfaceprojections or “spikes” (FIG. 10), characteristic for EBOV (Feldmann etal., 1996, Adv Virus Res 47, 1-52; Geisbert, et al., 1995, Virus Res 39,129-150). Immunogold staining of the VLPs with anti-Ebola GP antibodiesdemonstrated the identity of the spikes on the surface of the particlesas Ebola glycoprotein (FIG. 10B). VLPs for Marburg virus were alsogenerated in a similar manner.

In summary, a surrogate assay for the assembly and release of Ebolavirus that can be performed without the restrictions of biocontainmentlaboratories was established. This assay can be used for initialscreenings of agents that may inhibit Ebola virus budding.

3. Studies on the Role of TSG101 in Ebola Virus Life Cycle

We performed a series of biochemical studies to examine the involvementof the vaccuolar protein sorting (vps) protein TSG101 interaction withthe late domain of VP40 in EBOV assembly and release. A set of TSG101truncations C-terminally tagged with a Myc epitope were used for thesestudies. 293T cells were transfected with full length TSG101 and mutantstruncated at positions 140, 250 and 300 along with EBOV VP40. Cells werelysed after 48 h and subjected to immunoprecipitation with an anti-Mycantibody. As shown in FIG. 11, VP40 was coprecipitated with all TSG101proteins except for the 1-140 truncated mutant. Lack of association withthis mutant is consistent with the structural data that show thatresidues 141-145 make important contacts with an HIV Gag-derived PTAPpeptide (Pornillos et al., 2002, Nat Struct Biol 9, 812-7).Interestingly, the association of VP40 with 1-300 mutant of TSG101 wassignificantly stronger than with full length or 1-250 mutant (FIG. 11).The lower association of full length TSG101 can be attributed to thepresence of a PTAP motif in the C-terminal region of this molecule thatmay form an inter- or intramolecular association with the UEV domain ofTSG101. The dramatic reduction of interaction resulting from deletion ofamino acids 250-300 suggests that residues in this region may contributeto the binding to viral matrix proteins.

To confirm that TSG101 and VP40 associate directly and through the PTAPmotif, Far Western analysis was performed. Ebola VP40 (1-326) andtruncated (31-326) Ebola VP40 proteins as well as HA-tagged UEV domainof TSG101 were produced in bacteria. These proteins were electrophoresedon 4-20% gradient gel and electroblotted onto nitrocellulose membrane.Following blocking, the blot was incubated with a purified TSG 101protein (UEV), washed, and protein-protein interaction detected byenhanced chemiluminescence using an anti TSG101 antibody and HRP-labeledgoat anti rabbit as secondary antibody. As shown in FIG. 12, TSG 101interacts with the full length Ebola VP40 but not with the truncatedEbola VP40, confirming that the PTAP motif SEQ ID NO.: 7) at the Nterminus of VP40 plays a critical role in VP40-TSG101(UEV) interaction.An identical western blot developed with Ebola VP40 antibody coulddetect both the full length and the truncated VP40 showing the presenceof both the proteins on the blot.

4. Surface Plasmon Resonance Biosensor (SPR) Analysis of the EbolaVP40-TSG101 Interaction

A quantitative analysis of Ebola VP40 interacting with TSG101 wascarried out using SPR measurements. A biotinylated peptide(Bio-ILPTAPPEYME) containing 11 amino acid residues from the N terminusof the Ebola VP40 was immobilized on the streptavidine chip. PurifiedTSG101 protein that contains only the UEV domain was injected atdifferent concentrations (SEQ ID NO.: 9) (1, 2, 5, 20 uM) serially. Asseen in the FIG. 13, an interaction of moderate affinity between thepeptide and proteins can be detected. Based on the SPR data wecalculated a KD value of ˜2 μM for this interaction.

5. Incorporation of TSG101 in Ebola VLPs and Virions

To determine whether TSG101 is incorporated in EBOV VLPs, TSG101 (1-312)together with GP or GP+VP40 were expressed in 293T cells. VLPs wereimmunoprecipitated from supernatants using anti GP antibodies.Expression of TSG101 (1-312) resulted in a marked increase in VLPrelease, suggesting a positive role for TSG101 in VLP budding (FIG. 14A,Lane 4). In addition, TSG101 (1-312) was coimmunoprecipitated with ananti-GP antibody from the culture supernatants when expressed along withGP and VP40 (FIG. 14A, Lane 4). No TSG101 was found associated with GPwhen expressed in the absence of VP40 (FIG. 14A, lane 3), suggestingthat its association with GP was dependent on formation of VLPs.

Similar results were also obtained with full length TSG101. These datastrongly suggest that TSG101 is incorporated into VLPs and support thehypothesis that TSG101 plays a role in viral assembly and/or budding. Tofurther substantiate this finding we also analyzed inactivated, bandpurified, EBOV (iEBOV) for the presence of TSG101. 5 μg iEBOV wereanalyzed by immunoblotting for the presence of TSG101. As shown in FIG.6B, we found readily detectable levels of TSG101 in iEBOV, clearlydemonstrating the incorporation of TSG101 in Ebola virus.

6. Effect of Polyclonal Anti TSG101 Antibodies on Ebola Virus Release

The biochemical and VLP release data suggested that TSG101 is criticalfor the egress of Ebola virus. Therefore, the effects of polyclonalanti-TSG 101 antibodies “C” and “E” (that showed inhibitory effect onHIV, see Examples 1 and 2) were tested on the virus production in Helacells infected with Ebola Zaire-95 virus. Monolayers of Hela cells wereincubated with the virus at an MOI of 1 for 50 minutes, washed, and amedium containing an anti-TSG101 antibody or a control rabbit anti mouseantibody were added at 5 μg/ml. After 24 hours the supernatants wereharvested and the released viruses enumerated by plaque assay aspreviously described (Bavari et al., 2002, J Exp Med 195, 593-602). Asshown in FIG. 15, these antibodies partially inhibited the release ofvirions into Hela cell supernatant.

According to another set of embodiments, multiple and different types ofscreening assays to identify such compositions are provided, includingbut not necessarily restricted to, binding assays with TSG101 and/orassociated molecules/ligands, biochemical fractionation, chromatography,etc. For example, such assays may rely on the use of ELISA- or SurfacePlasmon Resonance (Biacore) techniques, etc. Such screening assays mayalso include (i) cell-based assays (e.g. to evaluate viral budding,release, or propagation in response to exposure to putativecompositions), such as flow cytometry, Tsg101 in situ localization orco-localization with other molecule(s)/ligand(s), labeling with markersof viral infection, viral titer assays, etc.; (ii) in silico assays(e.g., models or simulations based on known or anticipated structuralalgorithms of TSG101 interaction with other molecules, such as itscognate viral ligand(s)); or (iii) in vivo screens based on animal orcellular models for disease progression. Such methods may further relyon the use of arrays to simultaneously test multiple compositionsindividually and/or in duplicate.

TSG101 protein has been shown to play a role in intracellulartrafficking, transcriptional regulation, and cell cycle control. Duringviral infection, many viruses interact with TSG101 to carry out theirnormal infection cycle, and previous work has shown that mutating oraltering the expression level of TSG101 impedes the ability of viruses,such as HIV-1, to complete the infection cycle and produce infectiousparticles.

Recent work at FGI has shown that TSG101 protein, which is normallyrestricted to the cytoplasm of normal cells, is hijacked andmis-expressed at the cell surface during viral infection. The expressionand presentation of TSG101 on the surface of infected cells provides anew avenue for the development of novel tools and methods for thediagnosis and management of viral infections through identification andcharacterization of infected cells within a tissue. The effectiveness ofthis approach has been successfully confirmed through both laboratoryassays of viral infection in vitro and patient-derived samples whereviral infection arose naturally and not as a result of laboratoryintervention. Specifically, we have been able to demonstrate that TSG101antibodies identify cells that have been infected with HIV, influenza,RSV and multiple other viruses. Using HIV as an example, TSG101 surfaceexpression has been established using samples from naturally-infectedcells (where cells are infected with virus prior to the assay), fromlaboratory-based infection (where virus is added to the cultures in thelaboratory to achieve infection) and from horizontal infection (wherenaturally-infected cells release virus to infect exogenously-addedcells).

In the case of HIV, TSG101 surface exposure has also been observed innewly-infected cells, from patients with active HIV/AIDS disease andfrom long-term, asymptomatic survivors, who are HIV-positive. BeyondHIV, we expect that comparable surface exposure will also apply to manydifferent virus types that utilize TSG101 in their life cycle includingbut not necessarily restricted to Group I, III, IV, V, VI and VII virusfamilies as defined using the Baltimore Classification. This setincludes but is not restricted to HIV, RSV, Parainfluenza (PIV), andinfluenza. It is expected that Tsg101-based detection systems of thepresent invention may be applied to both veterinary and medical fields.

According to some embodiments of the present invention, methods andcompositions are provided for detection of Tsg101. Such detectingreagents for Tsg101 may be identified according to multiple anddifferent types of screening assays including, but not limited to, anyknown binding or co-fractionation assays or any known cell-basedsystems, such as microscopic analysis, flow cytometry, in situ staining,IHC, etc. Such detecting reagent(s) may be any directly or indirectlylabeled antibody, peptide, nucleic acid, or small molecule. For example,work at FGI has already demonstrated that TSG101 antibodies may be usedto selectively identify cells infected with virus.

The detecting agent could bind directly to any portion of TSG101.Alternatively, the detecting agent may bind to other molecules that areassociated with Tsg101, such as its viral ligand, either alone or incombination with Tsg101 itself. As an example, the UEV domain of TSG101could provide a target for broad-spectrum infection involving multipleand different types of viruses (e.g., HIV and influenza). Alternatively,a unique complex or epitope that is specifically useful for identifyinginfection caused by a particular virus may be used (e.g., to distinguishHIV from influenza). Differences between TSG101 conformations and/orcomplexes with different viruses may also be exploited to providespecific viral detection assays.

Many conventional formats for screening for the presence of TSG101 onthe surface of a cell, as a means of detecting and following viralinfection are offered. Conventional modalities will typically rely onthe detection of an agent that binds to TSG101, such as an antibody orantibody fragment, that in turn may be detected. The antibody may bear alabel or be otherwise detectable, or may itself be bound by a secondaryantibody that is in turn detectable. Such assays may be homogenous, allin solution, or in a more conventional format, the wells of a typicalreaction plate or other solid surface may be provided with theimmobilized capture antibody, the sample introduced, and the platewashed. Thereafter, a detectable antibody is introduced, which bindsselectively to a different epitope on the TSG101 target. The secondaryantibody is detectable through a variety of means, including afluorescent label, chemiluminesence, redox reactions giving electricalsignals, and other sensitive methods.

According to another set of embodiments of the present invention,compositions and methods are provided to distinguish among theinfections of viruses versus other pathogens (bacteria, fungi, etc).Such compositions and methods for detection of TSG101 could be usefulfor certain patient populations, especially pediatric or elderlypopulations, where early detection of viral infection could assist inthe course of therapy (e.g., whether to prescribe antibiotics).

According to another set of embodiments of the invention, TSG101 surfaceexposure could provide a means to monitor or track the progress of aviral infection. For example, HIV/AIDS is conventionally diagnosed usingassays that identify serum antibody production to the pathogen. Once apatient has sero-converted, the assay is no longer useful for measuringsubsequent disease progression, response to therapy, or other potentialdiagnostic, prognostic, or predictive outcomes. By directly observinginfected cells over time using TSG101-based detection assays describedherein, patient surveillance and disease management could be improved.For example, such TSG101-based assays could provide means for measuringthe response of a particular patient to his/her treatment regiment andthus be used to determine if a current course of disease management iseffective or if a new or altered course of disease management isrequired. This monitoring application is particularly useful, forexample, to determine if treatment should be continued or discontinuedwhen new and/or experimental treatments are being used.

According to yet another set of embodiments of the present invention,TSG101-based assays of viral infection could be specifically used todetermine eligibility for, or response to, specific therapies. Suchtherapies could include, for example, TSG101-based therapies such asantibodies, vaccines, small molecules and other therapeutic means thattarget TSG101 functions during viral infection. A linked diagnostic inthis case would be analogous to the use of certain detection assays foroncology applications (e.g., Herceptin and HercepTest) to determinepatient eligibility for a particular drug.

The following provides an exemplary method of the present invention forthe detection, diagnosis, or characterization of virally infected cellsusing a cell surface staining protocol:

-   -   1. Use about 0.1−0.5×10⁶ cell per well. Use round bottom tissue        culture 96 well plates    -   2. Trypsinize cells (unless they are non-adherent).    -   3. Spin at 1100 RPM for 5 min and aspirate supernatant    -   4. Resuspend in enough FACS buffer (PBS+1% BSA, filtered) to        obtain 1.0−5.0×10⁵ cells/100 ul (ex. One T75 flask resuspended        in 10 ml FACS buffer ˜100,000 cells/100 ul    -   5. Add 100 ul of cells to each well    -   6. Add primary antibody (amounts will vary depending on whether        it's purified Ab, supernatant, phage, etc).    -   7. Incubate at 4° C. for 40 min    -   8. Parafilm plate and spin at 1100 RPM for 5 min and aspirate        supernatant (wash aspirator with bleach when working with        infected cells)    -   9. Wash plate 3× with 200 ul FACS buffer, spinning plate and        aspirating supernatant each time    -   10. Incubate cells in 100 ul with Secondary Antibody (if primary        antibody is not conjugated) for 30 min. Where a Secondary        Antibody is used, it will bear a label or be otherwise        detectable.    -   11. Parafilm and spin plate at 1100 RPM for 5 min, aspirate        supernatant and add 200 ul FACS buffer. Repeat once more    -   12. Spin plate, aspirate supernatant and add 200 ul PBS to wells        (to remove proteins so they don't clog FACS machine)    -   13. Spin plate, aspirate supernatant    -   14. Fix with 200 ul of 1% PFA (paraformaldehyde-dilute 16%        solution in PBS). Wrap in foil and put in 4° C. until time to        read by Flow Cytometer.

Wells showing positive staining, or other evidence of retention of theprotein by the primary antibody are indicated to be infected, by reasonof the presence of TSG101 on the surface of those cells.

References Cited

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of the present invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims along with the full scope ofequivalents to which such claims are entitled.

What is claimed is:
 1. A method for determining if an intact_ mammaliancell is infected by an enveloped virus exhibiting a PPxP, PPxY, YxxL orPTAP motif, SEQ ID NOS.: 4-7, respectively, comprising: (a) contacting asurface of said intact mammalian cell with an antibody conjugate, saidantibody conjugate comprising an antibody that binds a TSG101 proteinconjugated with a label; and (b) detecting a cell having said labelattached, thereby identifying said cell as infected by said envelopedvirus.
 2. The method of claim 1, wherein said antibody binds theN-terminal or C-terminal region of said TSG101 protein.
 3. The method ofclaim 2, wherein said mammal is a human, and wherein said antibody bindsan epitope comprised in the amino acid region selected from the groupconsisting of VRETVNVITLYKDLKPVL (SEQ ID NO:2) and QLRALMQKARKTAGLSDLY(SEQ ID NO:3).
 4. The method of claim 1, wherein said virus is selectedfrom the group consisting of human immunodeficiency virus type I (HIV-1), human immunodeficiency virus type II (HIV-II), Marburg virus, Ebolavirus, and respiratory syncytial.
 5. The method of claim 1, wherein saidlabel is detected by fluorescence, chemilumiscence or colorimetricmethods.
 6. The method of claim 1, wherein said cell is bound first byan antibody to TSG101, and then said bound cell is contacted by saidantibody-conjugate.
 7. A method for determining if an intact mammaliancell is infected by an enveloped virus exhibiting a PPxP, PPxY, YxxL orPTAP motif, SEQ ID NOS.: 4-7, respectively, comprising: (a) contactingintact cells of a mammal with a first antibody that binds a TSG101protein on a surface of said cell when said cell is infected by anenveloped virus; and (b) contacting said cell bound by said firstantibody with a second antibody bearing a detectable label, wherein saidsecond antibody binds to said first antibody, wherein detection of saidlabel indicates the presence of a cell infected by an enveloped virus.8. The method of claim 7, wherein said first antibody that binds aTSG101 protein is immobilized prior to being contacted to said cells. 9.A method of monitoring the progress of a viral infection in a mammal,wherein said virus exhibits a PPxP, PPxY, YxxL or PTAP motif, SEQ IDNOS.: 4-7, respectively, comprising screening said mammal, over time,for the presence of virally infected cells, said screening processcomprising contacting intact cells of said mammal on each screen with anantibody which binds TSG101 and detecting the presence of said antibodybound to said cells, wherein the progress of said viral infection ismonitored by quantification of the number of virally infected cells insaid host on each screening.
 10. The method of claim 9, wherein saidantibody is detected directly or indirectly.